JP7625065B2 - Compositions and methods based on PMT engineering for producing tobacco plants and products with altered alkaloid levels - Google Patents

Description

INCORPORATION OF SEQUENCE LISTING This application claims the benefit of U.S. Provisional Application No. 62/703,775, filed July 26, 2018, and U.S. Provisional Application No. 62/848,159, filed May 15, 2019, both of which are incorporated by reference in their entireties. The Sequence Listing contained in the file entitled "P34620WO00_SL.txt" is 200,338 bytes (measured in MS-Windows), was created on July 25, 2019, and is submitted electronically herewith, and is incorporated by reference in its entirety.

FIELD The present disclosure provides genetic engineering of tobacco to modulate alkaloid and nicotine levels.

Background Nicotine is the predominant alkaloid, typically accounting for more than 90-95% of the total alkaloids in commercial tobacco cultivars. The remaining alkaloid fraction consists mainly of three additional alkaloids: nornicotine, anabasine, and anatabine. Tobacco plants with reduced nicotine levels have been obtained by modulating different nicotine biosynthetic genes and transcriptional regulators, but results have been variable and inconsistent. New technologies for reducing nicotine levels in tobacco leaves are needed.

SUMMARY The present disclosure provides tobacco plants having altered total alkaloid and nicotine levels and commercially acceptable leaf grades, their development through breeding or transgenic approaches, and the production of tobacco products from the tobacco plants.

In one aspect, the disclosure provides a tobacco plant or portion thereof comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant, when grown and processed under equivalent conditions, is capable of producing leaves comprising a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant that does not have the one or more mutant alleles.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

In a further aspect, the tobacco plant comprises one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

In a further aspect, the tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

In one aspect, the present disclosure provides a tobacco plant selected from the group consisting of a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, and a quintuple mutant, as listed in Tables 8A-8E.

In one aspect, the disclosure provides a tobacco plant listed in Tables 4A-4E or Table 10. In another aspect, the disclosure provides a progeny plant of a tobacco plant in Tables 4A-4E or Table 10, derived either by self-pollination or by crossing with another plant in Tables 4A-4E or Table 10.

In another aspect, the disclosure provides tobacco plants comprising various combinations of pmt mutant alleles listed in Tables 5A-5E or Tables 12A-12E resulting in a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, or a quintuple mutant. In one aspect, the disclosure provides tobacco plants comprising sequences of pmt mutant alleles selected from the group consisting of SEQ ID NOs: 21-200, 410-441, 474-505, 538-569, 602-633, and 666-697.

The present disclosure further provides cured tobacco, tobacco blends, and tobacco products comprising plant material derived from the disclosed tobacco plants, strains, varieties, or hybrids.
[The present invention 1001]
A tobacco plant or part thereof comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant, when grown and processed under equivalent conditions, is capable of producing leaves containing a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant not having the one or more mutant alleles.
[The present invention 1002]
The tobacco plant or part thereof of the present invention, wherein the tobacco plant comprises one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.
[The present invention 1003]
The tobacco plant or part thereof of the present invention, wherein the tobacco plant comprises one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.
[The present invention 1004]
The tobacco plant or part thereof of the present invention, wherein the tobacco plant comprises one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.
[The present invention 1005]
The tobacco plant or part thereof of the present invention, wherein the tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.
[The present invention 1006]
Any of the tobacco plants or parts thereof of 1001-1005 of the present invention, wherein the tobacco plant is capable of producing leaves containing nicotine levels that are 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% lower than the nicotine levels in leaves from a control tobacco plant not having the one or more mutant alleles when grown and processed under equivalent conditions.
[The present invention 1007]
The tobacco plant or part thereof of any of claims 1001-1006, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the total alkaloid level in leaves from the control tobacco plant when grown and processed under equivalent conditions.
[The present invention 1008]
The tobacco plant or part thereof of the present invention, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid level in leaves from the control tobacco plant when grown and processed under equivalent conditions.
[The present invention 1009]
1009. The tobacco plant or part thereof of any of claims 1001-1008, wherein the one or more mutant alleles comprise a mutation in a sequence region selected from the group consisting of a promoter, a 5'UTR, a first exon, a first intron, a second exon, a second intron, a third exon, a 3'UTR, a terminator, and any combination thereof.
[The present invention 1010]
The tobacco plant or part thereof of any of claims 1001 to 1009, wherein the one or more mutant alleles comprise one or more mutation types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof.
[The present invention 1011]
The one or more mutant alleles are:
The tobacco plant or part thereof of any of claims 1001 to 1010, which results in one or more of PMT protein truncation, a non-translated PMT gene transcript, a non-functional PMT protein, a premature stop codon in the PMT gene, and any combination thereof.
[The present invention 1012]
The tobacco plant or part thereof of any of claims 1001 to 1011, wherein the one or more mutant alleles comprise a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to a wild-type PMT gene.
[The present invention 1013]
The tobacco plant or part thereof of any of claims 1001-1012, wherein said one or more mutant alleles comprise a zygosity selected from the group consisting of homozygous, heterozygous, and heteroallelic.
[The present invention 1014]
The tobacco plant or part thereof of any of claims 1001 to 1012, wherein said one or more mutant alleles are homozygous or heteroallelic in at least one to five PMT genes.
[The present invention 1015]
The tobacco plant or part thereof of any of claims 1001 to 1012, wherein said one or more mutant alleles are homozygous or heteroallelic in at least four PMT genes.
[The present invention 1016]
The tobacco plant or part thereof of any of claims 1001 to 1012, wherein said one or more mutant alleles are homozygous or heterozygous in all five PMT genes.
[The present invention 1017]
The tobacco plant or part thereof according to any one of claims 1001 to 1016, wherein the at least two PMT genes are PMT1a and PMT3.
[The present invention 1018]
Any of the tobacco plants or parts thereof of the present invention, wherein the tobacco plant is capable of producing leaves containing a nicotine level selected from the group consisting of less than 0.15%, less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and less than 0.01%, by dry weight.
[The present invention 1019]
Any of the tobacco plants or parts thereof of the present invention, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level selected from the group consisting of less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, and less than 0.2%, by dry weight.
[The present invention 1020]
0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 ppm.
[The present invention 1021]
A population of any one of tobacco plants 1001 to 1020 of the present invention.
[The present invention 1022]
A dried tobacco material derived from any one of the tobacco plants of the present invention 1001 to 1020.
[The present invention 1023]
The dried tobacco material of the present invention 1022, which is produced by a drying process selected from the group consisting of hot air drying, air drying, flame drying, and sun drying.
[The present invention 1024]
Contains tobacco leaves,
the tobacco leaves exhibit reduced mold infection as compared to a control cured tobacco material from cultivar LA Burley 21;
The dried tobacco material of the present invention 1022.
[The present invention 1025]
A tobacco blend comprising the dried tobacco material of the present invention.
[The present invention 1026]
The tobacco blend of the present invention, wherein the dried tobacco material comprises at least about 10% by weight, at least 15% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, at least 35% by weight, at least 40% by weight, at least 45% by weight, at least 50% by weight, at least 55% by weight, at least 60% by weight, at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, or at least 95% by weight of the dried tobacco in the tobacco blend.
[The present invention 1027]
The tobacco blend of the present invention, wherein the dried tobacco material comprises at least about 10% by volume, at least 15% by volume, at least 20% by volume, at least 25% by volume, at least 30% by volume, at least 35% by volume, at least 40% by volume, at least 45% by volume, at least 50% by volume, at least 55% by volume, at least 60% by volume, at least 65% by volume, at least 70% by volume, at least 75% by volume, at least 80% by volume, at least 85% by volume, at least 90% by volume, or at least 95% by volume of the dried tobacco in the tobacco blend.
[The present invention 1028]
A tobacco product comprising the dried tobacco material of the present invention 1022.
[The present invention 1029]
The tobacco product of the present invention 1028 is selected from the group consisting of cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.
[The present invention 1030]
The tobacco product of the present invention, which is a smokeless tobacco product.
[The present invention 1031]
The tobacco product of the present invention 1030, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff.
[The present invention 1032]
A reconstituted tobacco comprising the dried tobacco material of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NOs:1-5 set forth exemplary genomic sequences for PMT1b, PMT1a, PMT2, PMT3, and PMT4, respectively, from the TN90 reference genome.

SEQ ID NOs:6-10 set forth exemplary cDNA sequences of PMT1b, PMT1a, PMT2, PMT3 and PMT4 from TN90, respectively.

SEQ ID NOs:11-15 set forth exemplary polypeptide sequences of PMT1b, PMT1a, PMT2, PMT3 and PMT4, respectively, from TN90.

SEQ ID NOs: 16-22 show exemplary guide RNA sequences.

SEQ ID NOs:23-200, 410-441, 474-505, 538-569, 602-633, and 666-697 describe exemplary edited pmt mutant sequences.

RNA expression of five PMT genes in TN90 roots. Nicotine levels in various low-alkaloid strains: CS15 (quintuple pmt knockout mutant CS15 in the NLM(Ph Ph) background, a PMT RNAi transgenic strain in the VA359 background) and low-nicotine KY171 ("LN KY171") cultivar (KY171 background carrying a nic1 and nic2 double mutation) compared to their respective normal-alkaloid control strains: NLM(Ph Ph), VA359, and KY171 backgrounds. Total alkaloid levels in various low-alkaloid strains: CS15, PMT RNAi, and LN KY171 compared to their respective normal-alkaloid control strains: NLM (Ph Ph), VA359, and KY171 backgrounds. Leaf yield in various low alkaloid lines: CS15, PMT RNAi, and LN KY171 compared to their respective normal alkaloid control lines: NLM (Ph Ph), VA359, and KY171 backgrounds. Leaf quality in various low-alkaloid lines: CS15, PMT RNAi, and LN KY171 compared to their respective normal-alkaloid control lines: NLM (Ph Ph), VA359, and KY171 backgrounds. Photographs showing mold growth on cured tobacco containing TN90LC (Figure 6A), LA BU21 (Figure 6B), TN90 containing an RNAi construct that downregulates PR50 (Figure 6C), TN90 containing an RNAi construct that downregulates PMT genes (Figure 6D), and TN90 containing edits to all five PMT genes (Figure 6E). See legend to FIG. 6A. See legend to FIG. 6A. See legend to FIG. 6A. See legend to FIG. 6A. Depiction of fungal infections observed in the strains investigated in Figures 6A-E.

Detailed Description Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Those skilled in the art will recognize the many methods that can be used to implement the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. For the purpose of this disclosure, the following terms are defined below.

Any references cited herein, including, for example, all patents and publications, are incorporated by reference in their entirety.

As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise clear from the context. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth by 10%.

As used herein, words such as "less than," "more than," "at least," "at most," "approximately," "below," "more than," and "about," when used in conjunction with a series of numerical values, modify each value in the series. For example, the phrase "less than 1%, 2%, or 3%" is equivalent to "less than 1%, less than 2%, or less than 3%."

As used herein, tobacco plants refer to plants derived from the species Nicotiana tabacum.

Nicotine biosynthesis in tobacco begins with the methylation of the polyamine putrescine to N-methylputrescine by the enzyme putrescine N-methyltransferase (PMT) using S-adenosyl-methionine as a cofactor. This is the step in which the precursor metabolite is involved in nicotine biosynthesis. The PMT enzyme is classified as EC 2.1.1.53 under the enzyme classification system. In Nicotiana tabacum, five genes encode putrescine N-methyltransferases, designated PMT1a, PMT1b, PMT2, PMT3, and PMT4. Table 1A lists the genomic DNA, cDNA, and protein sequences of these five PMT genes in TN90 plants. The present disclosure describes compositions and methods used to edit the PMT genes to produce pmt mutant plants with reduced nicotine levels while maintaining leaf quality.

As used herein, "PMT1b" or "PMT1b gene" refers to a genetic locus in tobacco that encodes a polypeptide having an exemplary amino acid sequence of TN90 set forth in SEQ ID NO:11.

As used herein, "PMT1a" or "PMT1a gene" refers to a genetic locus in tobacco that encodes a polypeptide having an exemplary amino acid sequence of TN90 set forth in SEQ ID NO:12.

As used herein, "PMT2" or "PMT2 gene" refers to a genetic locus in tobacco that encodes a polypeptide having an exemplary amino acid sequence of TN90 set forth in SEQ ID NO:13.

As used herein, "PMT3" or "PMT3 gene" refers to a genetic locus in tobacco that encodes a polypeptide having an exemplary amino acid sequence of TN90 set forth in SEQ ID NO:14.

As used herein, "PMT4" or "PMT4 gene" refers to a genetic locus in tobacco that encodes a polypeptide having an exemplary amino acid sequence of TN90 set forth in SEQ ID NO:15.

As used herein, a mutation refers to a genetic modification introduced into a gene to reduce, inhibit, or eliminate the expression or activity of a product encoded by the gene. Such a modification can be in any sequence region of the gene, such as the promoter, 5'UTR, exons, introns, 3'UTR, or terminator regions. In one aspect, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. As used herein, a "mutant allele" refers to an allele from a genetic locus where that allelic locus contains a mutation.

As used herein, "pmt mutant" refers to a tobacco plant that contains one or more mutations in one or more PMT genes. A pmt mutant can be a single mutant, a double mutant, a triple mutant, a quadruple mutant, or a quintuple mutant. As used herein, a single, double, triple, quadruple, or quintuple pmt mutant refers to a mutant having modifications in one, two, three, four, or five PMT genes, respectively. A pmt mutant can also be a combination of homozygous, heterozygous, or heteroallelic mutants in one or more PMT genes.

As used herein, gene or locus names are capitalized and italicized, e.g., PMT1a, PMT1b, PMT2, PMT3, and PMT4. Protein or polypeptide names are capitalized and not italicized, e.g., PMT1a, PMT1b, PMT2, PMT3, and PMT4. Variant names (to refer to common mutations in a gene or group of genes or to refer to specific variant alleles) are lowercase and italicized, e.g., pmt, pmt1a, pmt1b, pmt2, pmt3, and pmt4.

In one aspect, the disclosure provides a tobacco plant or portion thereof comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant, when grown and processed under comparable conditions, is capable of producing leaves comprising a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant not having the one or more mutant alleles. In one aspect, a single pmt mutant tobacco plant is provided. In another aspect, the single pmt mutant tobacco plant, when grown under similar growth conditions, comprises nicotine at a level that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant not having a single pmt mutation. In further aspects, the single pmt mutant tobacco plant contains nicotine at a level of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine level of a control plant not having the single pmt mutant when grown under similar growth conditions.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one aspect, a double pmt mutant tobacco plant is provided. In another aspect, the double pmt mutant tobacco plant comprises nicotine at a level less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant not having the double pmt mutation when grown under similar growth conditions. In further aspects, the double pmt mutant tobacco plants contain nicotine at levels between 1% and 5%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 95% of the nicotine levels of a control plant that does not have the double pmt mutation when grown under similar growth conditions.

In a further aspect, the tobacco plant comprises one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one aspect, a triple pmt mutant tobacco plant is provided. In another aspect, the triple pmt mutant tobacco plant comprises nicotine at a level less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant not having the triple pmt mutation when grown under similar growth conditions. In further aspects, the triple pmt mutant tobacco plants contain nicotine at levels between 1% and 5%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 95% of the nicotine levels of a control plant that does not have the triple pmt mutation when grown under similar growth conditions.

In another aspect, the tobacco plant comprises one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one aspect, a quadruple pmt mutant tobacco plant is provided. In another aspect, the quadruple pmt mutant tobacco plant comprises nicotine at a level less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant not having the quadruple pmt mutation when grown under similar growth conditions. In further aspects, the quadruple pmt mutant tobacco plants contain nicotine at levels between 1% and 5%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 95% of the nicotine levels of a control plant that does not have the quadruple pmt mutation when grown under similar growth conditions.

In a further aspect, the tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one aspect, a quintuple pmt mutant tobacco plant is provided. In another aspect, the quintuple pmt mutant tobacco plant comprises nicotine at a level less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant not having the quintuple pmt mutation when grown under similar growth conditions. In further aspects, the quintuple pmt mutant tobacco plant contains nicotine at a level of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine level of a control plant not having the quintuple pmt mutation when grown under similar growth conditions.

In one aspect, the tobacco plant is a single pmt mutant, double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant listed in Tables 8A-8E. In another aspect, the tobacco plant comprises one or more pmt mutant alleles listed in Tables 5A-5E and Tables 12A-12E. Each and every combination of the pmt mutant alleles listed in Tables 5A-5E and Tables 12A-12E is also provided to result in a single pmt mutant, double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant. Each of the mutant loci can be either homozygous or heterozygous, or can comprise a heteroallelic combination. In another aspect, the tobacco plant comprises a combination of pmt mutant genotypes as shown for each separate line listed in Tables 4A-4E and Table 10. In one aspect, the tobacco plant comprises a sequence of a pmt mutant allele selected from the group consisting of SEQ ID NOs: 21-200, 410-441, 474-505, 538-569, 602-633, and 666-697. In another aspect, the disclosure provides a double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant comprising a sequence of a pmt mutant allele selected from the group consisting of SEQ ID NOs: 21-200, 410-441, 474-505, 538-569, 602-633, and 666-697.

In one aspect, the tobacco plant, when grown and processed under comparable conditions, can produce leaves containing nicotine levels that are less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the nicotine levels of leaves from a control tobacco plant. In another aspect, the tobacco plant, when grown and processed under comparable conditions, can produce leaves containing nicotine levels that are 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine levels of a control tobacco plant.

In another aspect, the tobacco plant, when grown and processed under comparable conditions, can produce leaves containing a total alkaloid level that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the total alkaloid level of the leaves from a control tobacco plant. In another aspect, the tobacco plant, when grown and processed under comparable conditions, can produce leaves containing a total alkaloid level that is 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the total alkaloid level of the control tobacco plant.

In a further aspect, the tobacco plants, when grown and processed under comparable conditions, are capable of producing leaves containing total alkaloid levels that are less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid levels in leaves from control tobacco plants.

In one aspect, the mutant pmt allele comprises a mutation in a PMT sequence region selected from the group consisting of the promoter, 5'UTR, first exon, first intron, second exon, second intron, third exon, third intron, fourth exon, fourth intron, fifth exon, fifth intron, sixth exon, sixth intron, seventh exon, seventh intron, eighth exon, 3'UTR, terminator, and any combination thereof. In another aspect, the mutant pmt allele comprises a mutation in a PMT genomic sequence region listed in Tables 1D-1H.

In another aspect, the mutant pmt allele comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, and any combination thereof. In one aspect, the mutant pmt allele is a null allele or a knockout allele.

In one aspect, the mutant pmt allele results in one or more of PMT protein truncation, an untranslatable PMT gene transcript, a non-functional PMT protein, a premature stop codon in the PMT gene, and any combination thereof.

In another aspect, the mutant pmt allele comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to the wild-type PMT gene.

In one aspect, the pmt mutant comprises a zygosity selected from the group consisting of homozygous, heterozygous, and heteroallelic. In another aspect, the pmt mutant is homozygous or heteroallelic in at least one, two, three, four, or five PMT genes. In one aspect, the pmt mutant is homozygous or heteroallelic in at least four PMT genes. In another aspect, the pmt mutant is homozygous or heteroallelic in all five PMT genes. In another aspect, the pmt mutant comprises a mutation in PMT1a and PMT3.

In one aspect, the tobacco plant is capable of producing leaves containing a nicotine level selected from the group consisting of less than 0.15%, less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and less than 0.01% by dry weight.

In another aspect, the tobacco plant is capable of producing leaves containing a total alkaloid level selected from the group consisting of less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, and less than 0.2% by dry weight.

In further aspects, the tobacco plants are capable of producing dried leaves containing a total TSNA level of 2-0.05, 1.9-0.05, 1.8-0.05, 1.7-0.05, 1.6-0.05, 1.5-0.05, 1.4-0.05, 1.3-0.05, 1.2-0.05, 1.1-0.05, 1.0-0.05, 0.9-0.05, 0.8-0.05, 0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 ppm.

In one aspect, the tobacco plant, when dried, can produce leaves having a USDA grade index value selected from the group consisting of 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. In another aspect, the tobacco plant, when dried, can produce leaves having a USDA grade index value equivalent to that of a control plant when grown and dried under similar conditions, the control plant sharing essentially the same genetic background as the tobacco plant, except for the modification. In a further aspect, the tobacco plant, when dried, can produce leaves having a USDA grade index value of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index value of the control plant when grown under similar conditions, the control plant sharing essentially the same genetic background as the tobacco plant, except for the modification. In a further aspect, the tobacco plant, when dried, can produce leaves having a USDA grade index value of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA grade index value of the control plant. In a further aspect, the tobacco plant, when dried, can produce leaves having a USDA grade index value of 70% to 125%, 75% to 120%, 80% to 115%, 85% to 110%, or 90% to 100% of the USDA grade index value of the control plant.

In one aspect, the tobacco plant contains nicotine at a level that is less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of a control plant when grown under similar growth conditions, and the control plant shares essentially the same genetic background as the tobacco plant, except for the modification.

In a further aspect, the tobacco plant comprises one or more pmt mutant alleles and further comprises a transgene or mutation that directly inhibits expression or activity of one or more genes encoding a product selected from the group consisting of MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP), and a MATE transporter.

In one aspect, the tobacco plant comprises one or more pmt mutant alleles and further comprises a mutation in an ERF gene at the Nic2 locus. In one aspect, the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. See Shoji et al., Plant Cell, (10): 3390-409 (2010); and Kajikawa et al.,, Plant physiol. 2017, 174: 999-1011. In one aspect, the tobacco plant further comprises one or more mutations in ERF189, ERF115, or both.

In one aspect, the tobacco plant comprises one or more qpt mutant alleles and further comprises a mutation in an ERF gene at the Nic1 locus (or Nic1b locus, as found in PCT/US2019/013345, filed Jan. 11, 2019, published as WO/2019/140297). See also WO/2018/237107. In one aspect, the tobacco plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. Kajikawa et al., Plant Physiol. 2017, 174:999-1011. In one aspect, the tobacco plant further comprises one or more mutations in one or more, two or more, three or more, four or more, five or more, or all six genes selected from the group consisting of ERFnew, ERF199, ERF19, ERF29, ERF210, and ERF91L2.

In one aspect, the disclosure further provides a pmt mutant tobacco plant or portion thereof comprising a nicotine or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.025%, less than 0.01%, and less than 0.005%, wherein the tobacco plant, when dried, is capable of producing leaves having a USDA grade index value of 50 or greater, 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater. In another aspect, such pmt mutant tobacco plants are capable of producing leaves containing nicotine levels of less than 0.02% and, when dried, having a USDA grade index value of 70 or greater. In a further aspect, such tobacco plants are capable of producing leaves containing nicotine levels of less than 0.01% and, when dried, having a USDA grade index value of 70 or greater.

In one aspect, the disclosure also provides a tobacco plant or portion thereof comprising a non-transgenic mutation, the non-transgenic mutation reducing the nicotine level or total alkaloid level of the tobacco plant to less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of a control plant when grown under similar growth conditions, the tobacco plant being capable of producing leaves, when dried, having a USDA grade index value equivalent to the USDA grade index value of the control plant, the control plant sharing essentially the same genetic background as the tobacco plant except for the non-transgenic mutation.

In one aspect, the tobacco plant comprises a pmt mutation introduced by an approach selected from the group consisting of random mutagenesis and targeted mutagenesis. In another aspect, the pmt mutation is introduced by a targeted mutagenesis approach selected from the group consisting of meganucleases, zinc finger nucleases, TALENs, and CRISPRs.

Unless otherwise specified, measurements of alkaloid or nicotine levels (or another leaf chemistry or characteristic assessment) or leaf grade index values referred to herein for a tobacco plant, variety, cultivar, or strain refer to average measurements, including, for example, the average of multiple leaves from a single plant, or the average measurement from a population of tobacco plants derived from a single variety, cultivar, or strain.

Unless otherwise stated, nicotine or alkaloid levels (or another leaf chemistry or characterization) of tobacco plants are measured after topping in pooled leaf samples collected from leaf numbers 3, 4, and 5 after topping. As used herein, whenever a comparison between leaves from two plants (e.g., mutant and control plants) is mentioned, leaves from the same or equivalent leaf position(s) and developmental stage(s) are intended, from which effects due to genotypic differences, and not from other factors, can be demonstrated. By way of illustration, leaf 3 of a wild-type control plant is intended as a reference point for comparison with leaf 3 of a pmt mutant plant. In one aspect, a tobacco plant containing at least one pmt mutation is compared to a control tobacco plant of the same tobacco variety.

The nicotine or alkaloid levels (or other leaf chemistry or characteristics) of the tobacco plant can also be measured by alternative methods. In one embodiment, the nicotine or alkaloid levels (or other leaf chemistry or characteristics) of the tobacco plant are measured after pruning in the leaves with the highest levels of nicotine or alkaloids (or other leaf chemistry or characteristics). In one embodiment, the nicotine or alkaloid levels of the tobacco plant are measured after pruning in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 leaves. In another aspect, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plants are measured after deflowering in two or more pools of consecutive leaf numbers selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In another aspect, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plants are measured after deflowering in leaves of a number of leaves selected from the group consisting of: 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30. In another aspect, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of tobacco plants are measured after pruning in two or more pools of leaves with a number of leaves selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30. In another aspect, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of tobacco plants are measured after pruning in three or more pools of leaves with a number of leaves selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30.

As used herein, leaf numbering is based on the location of the leaf on the tobacco stem, with leaf number 1 after pruning being the youngest leaf (top) and the highest leaf number being the oldest leaf (bottom).

The population of tobacco plants or collection of tobacco leaves for determining the average measurement (e.g., alkaloid or nicotine levels or leaf grading) can be of any size, e.g., 5, 10, 15, 20, 25, 30, 35, 40, or 50. Standard industry-accepted protocols are followed for determining the average measurement or grading index value.

As used herein, "deflowering" refers to removing the shoot apex, including the shoot apical meristem (SAM), flowers, and up to some adjacent leaves, when the tobacco plant is near plant maturity and at the beginning of reproductive growth. Typically, tobacco plants are deflowered at the button stage (shortly after the flowers begin to appear). For example, greenhouse or field-grown tobacco plants can be deflowered when 50% of the plants have at least one flowering. Deflowering tobacco plants causes a loss of apical dominance and induces increased alkaloid production.

Unless otherwise indicated, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plant are measured two weeks after deflowering. Alternatively, other time points can be used. In one aspect, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plant are measured about 1, 2, 3, 4, or 5 weeks after deflowering. In another aspect, the nicotine, alkaloid, or polyamine levels (or another leaf chemistry or characteristic) of the tobacco plant are measured about 3, 5, 7, 10, 12, 14, 17, 19, or 21 days after deflowering.

As used herein, "similar growing conditions" or "equivalent growing conditions" refers to similar environmental conditions and/or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrients (e.g., nitrogen and phosphorus). Agronomic practices include, for example, seeding, clipping, undercutting, transplanting, topping, and suckering. Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds. , Blackwell Publishing, Oxford (1999), pp 70-103, Chapters 4B and 4C.

An "alkaloid" is a complex nitrogen-containing compound that occurs naturally in plants and has pharmacological effects in humans and animals. "Nicotine" is the major naturally occurring alkaloid in commercial cigarette tobacco, accounting for approximately 90% of the alkaloid content in Nicotiana tabacum. Other major alkaloids in tobacco include cotinine, nornicotine, myosmine, nicotyrine, anabasine, and anatabine. Minor tobacco alkaloids include nicotine-n-oxide, N-methylanatabine, N-methylanabasine, pseudooxynicotine, 2,3 dipyridyl, and others.

Alkaloid levels can be assayed by methods known in the art, for example, by gas-liquid chromatography, high performance liquid chromatography, radioimmunoassay, and enzyme-linked immunosorbent assay based quantification. For example, nicotinic alkaloid levels can be measured by GC-FID based on CORESTA Recommended Method No. 7, 1987 and ISO standard (ISO TC 126N 394 E. For a method using gas-liquid chromatography equipped with a capillary column and FID detector, see Hibi et al., Plant Physiology 100:826-35 (1992).

Unless otherwise noted, alkaloid and nicotine levels are measured using methods according to those defined in CORESTA Method No. 62, Determination of Nicotine in Cigarettes and Tobacco Products by Gas Chromatographic Analysis, February 2005, and the Centers for Disease Control and Prevention Protocol for the Analysis of Nicotine, Total Moisture, and pH in Smokeless Tobacco Products, published in Federal Register Vol. 64, No. 55, March 23, 1999 (and amended Vol. 74, No. 4, January 7, 2009). Alternatively, tobacco total alkaloids can be measured using a segmented flow colorimetric method developed for the analysis of tobacco samples adapted by Skallar Instrument Co (West Chester, PA) and described in Collins et al., Tobacco Science 13:79-81 (1969). Briefly, tobacco samples can be dried, ground, and extracted prior to analysis of total alkaloids and reducing sugars. The method then uses acetic acid/methanol/water extraction and charcoal for decolorization. Total alkaloid determination is based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines to form a colored complex that is measured at 460 nm. Unless otherwise stated, total alkaloid or nicotine levels presented herein are on a dry weight basis (e.g., percentage of total alkaloids or percentage of nicotine).

In one embodiment, the tobacco plant is at about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 26%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100 %, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9%. In another aspect, the tobacco plant comprises, on a dry weight basis, about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, 1.4% to 1.5%, 1.5% to 1. .6%, 1.6%-1.7%, 1.7%-1.8%, 1.8%-1.9%, 1.9%-2%, 2%-2.1%, 2.1%-2.2%, 2.2%-2.3%, 2.3%-2.4%, 2.4%-2.5%, 2.5%-2.6%, 2.6%-2.7%, 2.7%-2.8%, 2.8%-2.9%, 2.9%-3%, 3%-3.1%, 3.1%-3.2%, 3.2%-3.3%, 3.3%-3.4%, 3.4%-3.5%, and 3.5%-3.6%. In further aspects, the tobacco plant comprises an average nicotine level or total alkaloid level selected from the group consisting of about 0.01%-0.1%, 0.02%-0.2%, 0.03%-0.3%, 0.04%-0.4%, 0.05%-0.5%, 0.75%-1%, 0.1%-1.5%, 0.15%-2%, 0.2%-3%, and 0.3%-3.5% on a dry weight basis.

The present disclosure also provides tobacco plants with altered nicotine levels that do not adversely affect other tobacco traits, such as leaf grade index values. In one aspect, low-nicotine or nicotine-free tobacco varieties provide cured tobacco of commercially acceptable grades. Tobacco grades are evaluated based on factors including, but not limited to, leaf position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to leaf strength and color depth and brilliance), hygroscopicity (the ability of tobacco leaves to absorb and retain moisture from the environment), and green tint or color cast. Leaf grades can be determined, for example, using official standard grades published by the Agricultural Marketing Service of the United States Department of Agriculture (7 U.S.C. § 511). See, for example, the Official Standard Grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93) (55 F.R. 40645), effective November 5, 1990; the Official Standard Grades for Flue-Cured Tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92) (54 F.R. 7925), effective March 27, 1989; the Official Standard Grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41) (29 F.R. 16854), effective January 8, 1965; the Official Standard Grades for Ohio Cigar-Leaf Tobacco (U.S. Type 41) (29 F.R. 16854), effective December 8, 1963; Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 42, 43, and 44) (28 F.R. 11719 and 28 F.R. 11926), Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective November 20, 1969, Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective April 1971, Official Standard Grades for Georgia and Florida Shade-Grown Cigar Wrapper Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective April 1971, See Official Standard Grades of Florida Shade-Grown Cigar-Wrapper Tobacco (US Type 62). USDA Grade Index values may be determined according to industry accepted grading indices. See, e.g., Bowman et al, Tobacco Science, 32:39-40 (1988), Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index), and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated by reference in their entirety). In one aspect, the USDA Grade Index is a numerical representation of the federal grade from 0 to 100, which is a weighted average of all leaf positions. The higher the grade index, the higher the quality. Alternatively, leaf grade can be determined via hyperspectral imaging. See, e.g., WO 2011/027315 (published March 10, 2011, and incorporated by reference in its entirety).

In one aspect, the tobacco plants provided herein contain similar levels of one or more tobacco aroma compounds when grown under similar growth conditions, as compared to a control tobacco plant. In another aspect, the tobacco plants provided herein contain similar levels of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, labdenoids, cembrenoids, sugar esters, and reducing sugars, as compared to a control tobacco plant when grown under similar growth conditions.

As used herein, tobacco aroma compounds are compounds associated with the flavor and aroma of tobacco smoke. These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, cembrenoids and labdenoids, diterpenes, and sugar esters. The concentration of tobacco aroma compounds can be measured by any known metabolite profiling method in the art, including, but not limited to, gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry. See The Handbook of Plant Metabonomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).

As used herein, a "reducing sugar(s)" is any sugar (monosaccharide or polysaccharide) that has a free or potentially free aldehyde or ketone group. Glucose and fructose act as nicotine buffers in cigarette smoke by reducing the pH of the smoke and effectively reducing the amount of "free" unprotonated nicotine. Reducing sugars balance the flavor of the smoke, for example, by modifying the sensory impact of nicotine and other tobacco alkaloids. An inverse relationship between sugar content and alkaloid content has been reported between tobacco varieties, within the same variety, and within the same plant line due to growing conditions. Reduction of sugar levels can be measured using a segmented flow colorimetric method developed for the analysis of tobacco samples adapted by Skalar Instrument Co (West Chester, PA) and described in Davis, Tobacco Science 20:139-144 (1976). For example, a sample is dialyzed against a sodium carbonate solution. Copper neocuproine is added to the sample and the solution is heated. The copper neocuproine chelate is reduced in the presence of sugar, resulting in a colored complex that is measured at 460 nm.

In one aspect, the tobacco plant contains one or more non-naturally occurring mutant alleles at one or more PMT loci that reduce or eliminate PMT enzyme activity from one or more PMT loci. In one aspect, these mutant alleles result in lower nicotine levels. The mutant pmt alleles can be introduced by any method known in the art, including random mutagenesis or targeted mutagenesis approaches.

Such mutagenesis methods include, but are not limited to, ethyl methylsulfate (EMS) (Hildering and Verkerk, In, The use of induced mutations in plant breeding. Pergamon press, pp 317-320, 1965), or UV irradiation, X-rays, and fast neutron irradiation (see, e.g., Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971, and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3.sup.rd) ed), 1987), transposon tagging (Fedoroff et al., 1984; U.S. Pat. Nos. 4,732,856 and 5,013,658), and seed treatment with T-DNA insertion techniques (Hoekema et al., 1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis consists of chemically induced random point mutations along the length of the genome. Rapid neutron mutagenesis consists of exposing seeds to neutron irradiation, which causes large deletions through double-stranded DNA breaks. Transposon tagging involves the insertion of a transposon within an endogenous gene to reduce or eliminate expression of the gene. Types of mutations that can be present in tobacco genes include, for example, point mutations, deletions, insertions, duplications, and inversions. Desirably, such mutations are in the coding regions of the tobacco gene, although mutations in promoter regions and introns, or untranslated regions of the tobacco gene, may also be desirable.

In addition, a rapid and automatable method for screening for chemically induced mutations, TILLING (targeted local disruption of the genome), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the present disclosure. See McCallum et al. (2000) Nat. Biotechnol. 18:455-457. Mutations that affect gene expression or prevent gene function can be determined using methods well known in the art. Insertional mutations in gene exons usually result in null mutants. Mutations in conserved residues can be particularly effective at disrupting protein function. In one aspect, the tobacco plant contains a nonsense (e.g., stop codon) mutation in one or more of the PMT genes described herein.

It should be understood that when identifying a mutation, the endogenous reference DNA sequence should be from the same tobacco variety. For example, if the modified tobacco plant containing the mutation is from variety TN90, the endogenous reference sequence should be the endogenous TN90 sequence, not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if the modified tobacco cell containing the mutation is a TN90 cell, the endogenous reference sequence should be the endogenous TN90 sequence, not a homologous sequence from a tobacco cell from a different tobacco variety (e.g., K326).

In one aspect, the present disclosure also provides tobacco strains with altered nicotine levels while maintaining commercially acceptable leaf quality. The strains can be produced by introducing mutations into one or more PMT genes via precise genome engineering techniques, such as transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, CRISPR/Cpf1 systems, CRISPR/Csm1 systems, and combinations thereof (see, e.g., U.S. Patent Application Publication No. 2017/0233756). See, e.g., Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013).

Screening and selection of mutagenized tobacco plants can be performed through any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for detection of polynucleotides, Northern blots, RNase protection, primer extension, RT-PCR amplification for detection of RNA transcripts, Sanger sequencing, next generation sequencing techniques (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting enzymatic or ribozyme activity of polypeptides and polynucleotides, as well as protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing the cited techniques are all known.

In one aspect, the tobacco plant or plant genome provided herein is mutated or edited by a nuclease selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas9 nuclease, a CRISPR/Cpf1 nuclease, or a CRISPR/Csm1 nuclease.

As used herein, "editing" or "genome editing" refers to targeted mutagenesis of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides of an endogenous plant genomic nucleic acid sequence, or removal or replacement of an endogenous plant genomic nucleic acid sequence. In one aspect, the edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity with the endogenous nucleic acid sequence. In one aspect, the edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-10, and fragments thereof. In another aspect, the edited nucleic acid sequence provided has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 11-15.

Meganucleases, ZFNs, TALENs, CRISPR/Cas9, CRISPR/Csm1 and CRISPR/Cpf1 induce double-stranded DNA breaks at the target site of the genomic sequence, which are then repaired by the natural processes of homologous recombination (HR) or non-homologous end joining (NHEJ). Sequence modifications then occur at the break site, which may include deletions or insertions resulting in gene disruption in the case of NHEJ, or integration of the donor nucleic acid sequence by HR. In one aspect, the method provided comprises editing a plant genome with a provided nuclease, and mutating at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten nucleotides in the plant genome through HR with a donor polynucleotide. In one aspect, the mutations provided are caused by genome editing using a nuclease. In another embodiment, the mutations provided are caused by non-homologous end joining or homologous recombination.

Meganucleases, commonly identified in microorganisms, are unique enzymes with high activity and long recognition sequences (>14 bp) that result in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14-40 bp). Engineering meganucleases can be more challenging than engineering ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined within a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and have improved nuclease activity.

ZFNs are synthetic proteins consisting of engineered zinc finger DNA binding domains fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be designed to cleave double-stranded DNA of almost any length due to the modification of the zinc finger DNA binding domain. ZFNs form dimers from monomers consisting of the non-specific DNA cleavage domain of the FokI endonuclease fused to a zinc finger array engineered to bind to the target DNA sequence.

The DNA binding domain of a ZFN typically consists of an array of three to four zinc fingers. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger ∞-helix, which contribute to site-specific binding to the target DNA, can be altered and customized to fit specific target sequences. Other amino acids form a consensus scaffold to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.

The FokI nuclease domain requires dimerization to cleave DNA, and therefore requires two ZFNs with their C-terminal regions to bind to DNA strands (separated by 5-7 bp) on opposite sides of the cleavage site. A ZFN monomer can cleave a target site if the two ZF binding sites are palindromic. As used herein, the term ZFN is broad and includes monomeric ZFNs that can cleave double-stranded DNA without the assistance of another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs engineered to function together to cleave DNA at the same site.

Without being limited to any scientific theory, the DNA-binding specificity of zinc finger domains can in principle be re-engineered using one of a variety of methods, so customized ZFNs could theoretically be constructed to target almost any gene sequence. Commonly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

TALENs are artificial restriction enzymes generated by fusing a transcription activator-like effector (TALE) DNA binding domain to a FokI nuclease domain. When each member of a TALEN pair binds to a DNA site adjacent to the target site, the FokI monomer dimerizes and causes a double-stranded DNA break at the target site. As used herein, the term TALEN is broad and includes monomeric TALENs that can cleave double-stranded DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that function together to cleave DNA at the same site.

Transcription activator-like effectors (TALEs) can be engineered to bind virtually any DNA sequence. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas. Xanthomonas pathogens secrete TALEs into host plant cells during infection. TALEs translocate to the nucleus and recognize and bind to specific DNA sequences in the promoter regions of specific genes in the host genome. TALEs have a central DNA-binding domain consisting of 13-28 repeats of 33-34 amino acid monomers. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat variable dimers (RVDs). The amino acid pairs NI, NG, HD, and NN of the RVD preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and the regulation of the RVD can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing the appropriate RVD.

In addition to the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and activity. The FokI domain functions as a dimer and requires two constructs with unique DNA binding domains to a site in the target genome with appropriate orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN binding sites, are parameters for achieving high levels of activity.

The relationship between the amino acid sequence of the TALE binding domain and DNA recognition allows for designable proteins. Software programs such as DNA Works can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. See Doyle et al., Nucleic Acids Research (2012) 40:W117-122, Cermak et al., Nucleic Acids Research (2011). 39:e82 and tale-nt. cac. cornell. edu/about.

The CRISPR/Cas9 system, CRISPR/Csm1, or CRISPR/Cpf1 system is an alternative to the FokI-based methods ZFN and TALEN. The CRISPR system is based on an RNA-guided engineered nuclease that uses complementary base pairing to recognize DNA sequences at target sites.

The CRISPR/Cas9, CRISPR/Csm1, and CRISPR/Cpf1 systems are part of the adaptive immune system of bacteria and archaea, protecting them from invading nucleic acids such as viruses by cleaving foreign DNA in a sequence-dependent manner. Immunity is acquired by the incorporation of a short piece of invading DNA, known as a spacer, between two adjacent repeats at the proximal end of the CRISPR locus. The spacer-containing CRISPR array is transcribed during the subsequent encounter with the invading DNA and processed into a ~40 nt long small interfering CRISPR RNA (crRNA), which in combination with the transactivating CRISPR RNA (tracrRNA) activates and guides the Cas9 nuclease, which cleaves the homologous double-stranded DNA sequence, known as the protospacer, in the invading DNA. A prerequisite for cleavage is the presence of a conserved protospacer adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5-NGG-3, and less frequently NAG. Specificity is provided by a so-called "seed sequence" about 12 bases upstream of the PAM, which must match between the RNA and the target DNA. Cpf1 and Csm1 act in a similar manner to Cas9, but Cpf1 and Csm1 do not require tracrRNA.

In yet another aspect, the tobacco plants provided herein comprise one or more pmt mutations and further comprise one or more mutations at one or more loci encoding nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) that confer a reduced amount of nornicotine compared to a control plant lacking one or more mutations at one or more loci encoding nicotine demethylase (see U.S. Pat. Nos. 8,319,011, 8,124,851, 9,187,759, 9,228,194, 9,228,195, and 9,247,706). In one aspect, the described tobacco plants further comprise reduced nicotine demethylase activity compared to a control plant when grown and dried under comparable conditions.

In one aspect, the pmt mutant tobacco plant further comprises a mutation that, when grown and processed under equivalent conditions, is capable of producing leaves containing anabasine levels that are less than the anabasine levels of leaves from a wild-type control tobacco plant. In another aspect, the pmt mutant tobacco plant further comprises a mutation that, when grown and processed under equivalent conditions, is capable of producing leaves containing anabasine levels that are less than 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the anabasine levels of leaves from a wild-type control tobacco plant.

In one aspect, the pmt mutant tobacco plant comprises an additional mutation capable of producing leaves containing anatabine levels that are reduced by 2-fold or more when grown and processed under equivalent conditions, compared to leaves from a control tobacco plant. In another aspect, the pmt mutant tobacco plant comprises an additional mutation capable of producing leaves containing anatabine levels that are reduced by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 13-fold when grown and processed under equivalent conditions, compared to leaves from a wild-type control tobacco plant. In one aspect, the mutation that provides lower levels of anatabine is a mutation described in U.S. Patent Publication Nos. 2014/0283165 and 2016/0010103. In another aspect, the pmt mutant further comprises a mutation in a quinolate phosphoribosyltransferase (QPT) or quinolinate synthase (QS) gene. In a further aspect, the pmt mutant plant further comprises an introduced gene or mutation that suppresses expression or activity of the QPT or QS gene.

In one aspect, the pmt mutant tobacco plant further comprises a mutation capable of providing a nornicotine level that is 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, or 35% lower than the leaf nornicotine level from a wild-type control tobacco plant when grown and processed under equivalent conditions.

In one aspect, pmt mutant tobacco plants are capable of producing dried leaves containing total N-nitrosonornicotine (NNN) levels of less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05 ppm.

In another aspect, pmt mutant tobacco plants are capable of producing dried leaves containing a total NNN level of 2-0.05, 1.9-0.05, 1.8-0.05, 1.7-0.05, 1.6-0.05, 1.5-0.05, 1.4-0.05, 1.3-0.05, 1.2-0.05, 1.1-0.05, 1.0-0.05, 0.9-0.05, 0.8-0.05, 0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 parts per million (ppm).

In one aspect, pmt mutant tobacco plants are capable of producing cured leaves containing total nicotine-derived nitrosamine ketone (NNK) levels of less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05 ppm.

In another aspect, pmt mutant tobacco plants are capable of producing dried leaves containing total NNK levels of 2-0.05, 1.9-0.05, 1.8-0.05, 1.7-0.05, 1.6-0.05, 1.5-0.05, 1.4-0.05, 1.3-0.05, 1.2-0.05, 1.1-0.05, 1.0-0.05, 0.9-0.05, 0.8-0.05, 0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 ppm.

In one aspect, the pmt mutant tobacco plant further comprises a mutation or transgene that provides an increased level of one or more antioxidants. In another aspect, the pmt mutant tobacco plant further comprises a genetic modification in an endogenous gene, further comprising an increased level of one or more antioxidants in the cured leaves compared to a control cured tobacco leaf lacking the genetic modification, the endogenous gene encoding an antioxidant biosynthetic enzyme, an antioxidant regulatory transcription factor, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In a further aspect, the pmt mutant tobacco plant further comprises a transgene, further comprising an increased level of one or more antioxidants in the cured leaves compared to a control cured tobacco leaf lacking the transgene, the transgene encoding or directly regulating an antioxidant biosynthetic enzyme, an antioxidant regulatory transcription factor, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In one aspect, the pmt mutant tobacco plant further comprises a transgene or cisgenic construct expressing one or more genes selected from the group consisting of AtPAP1, NtAN2, NtAN1, NtJAF13, NtMyb3, chorismate mutase, and arogenate dehydrotase (ADT). In another aspect, the pmt mutant tobacco plant further comprises one or more transgenes or genetic modifications to increase antioxidants or decrease one or more TSNAs, as described in WIPO Publication No. 2018/067985 or U.S. Publication No. 2018/0119163.

In one aspect, the described tobacco plants are modified tobacco plants. As used herein, "modified" in the context of a plant refers to a plant that contains genetic alterations that have been introduced for a particular purpose and beyond natural polymorphisms.

In one aspect, the described tobacco plants are cisgenic plants. As used herein, "cisgenesis" or "cisgenic" refers to the genetic modification of a plant, plant cell, or plant genome in which all components (e.g., promoters, donor nucleic acids, selection genes) have only plant origin (i.e., no non-plant origin components are used). In one aspect, the provided plants, plant cells, or plant genomes are cisgenic. The provided cisgenic plants, plant cells, and plant genomes can lead to ready-to-use tobacco lines. In another aspect, the provided tobacco plants are free of non-tobacco genetic material or sequences.

As used herein, "gene expression" or expression of a gene refers to the biosynthesis or production of a gene product, including the transcription and/or translation of the gene product.

In one aspect, the provided tobacco plant comprises one or more pmt mutations and further comprises reduced expression or activity of one or more genes involved in nicotine biosynthesis or nicotine transport. Genes involved in nicotine biosynthesis include, but are not limited to, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), quinolinic acid phosphoribosyltransferase (QPT), and S-adenosylmethionine synthase (SAMS). Nicotine synthase, which catalyzes the condensation step between nicotinic acid derivatives and the methylpyrrolinium cation, has not been elucidated, although two candidate genes (A622 and NBB1) have been proposed. See US2007/0240728A1 and US2008/0120737A1. A622 encodes an isoflavone reductase-like protein. In addition, several transporters may be involved in the permeation of nicotine. A transporter gene named MATE has been cloned and characterized (Morita et al., PNAS 106:2447-52 (2009)).

In one aspect, the provided tobacco plants comprise one or more pmt mutations and further comprise reduced levels of mRNA, protein, or both of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1, as compared to a control tobacco plant. In another aspect, the provided tobacco plants comprise one or more pmt mutations and further comprise a transgene that directly suppresses expression of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. In another aspect, the provided tobacco plants comprise one or more pmt mutations and further comprise a transgene or mutation that suppresses expression or activity of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1.

In one aspect, the tobacco plant provided is from a tobacco variety selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark air-cured tobacco, dark flue-cured tobacco, Galpao tobacco, and Oriental tobacco. In another aspect, the tobacco plant provided is from a tobacco variety selected from the group consisting of Burley tobacco, Maryland tobacco, and dark tobacco.

In one aspect, the provided tobacco plant is in the flue-cured tobacco background or exhibits one or more flue-cured tobacco characteristics described herein. Flue-cured tobacco (also called Virginia tobacco or light tobacco) represents approximately 40% of the world's tobacco production. Flue-cured tobacco is often referred to as "light tobacco" because it turns golden to deep orange during curing. Flue-cured tobacco has a light and distinctive aroma and taste. Flue-cured tobacco is generally high in sugar and low in oil. The major growing countries of flue-cured tobacco are Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, the provided low alkaloid or low nicotine tobacco plants or seeds are selected from the group consisting of CC13, CC27, CC33, CC37, CC65, CC67, CC700, GF318, GL338, GL368, GL939, K346, K399, K326, NC102, NC196, NC291, NC297, NC299, NC471, NC55, NC In the context of the present invention, a flue-cured tobacco is selected from the group consisting of: NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety essentially derived from any one of the aforesaid varieties. In another aspect, the low alkaloid or low nicotine tobacco plants or seeds provided are selected from the group consisting of Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL939, K149, K326, K340, K346, K358, K394, K399, K730, NC27NF, NC37NF, NC55, NC60, NC71, NC72, NC82, NC95, NC297, NC606, NC729, NC2326, McNair 373, McNair 944, Ox207, Ox414NF, Reams 126, Reams 713, Reams 744, RG8, RG11, RG13, RG17, RG22, RG81, RGH4, RGH51, Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108, Speight G-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va116, Va182, and any variety essentially derived from any one of the aforesaid varieties. See WO 2004/041006 A1. In a further aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or strain is on any hot air cured background selected from the group consisting of K326, K346, and NC196.

In one aspect, the provided tobacco plants are in the context of air-cured tobacco or exhibit one or more of the characteristics of air-cured tobacco described herein. Air-cured tobaccos include burley, Maryland, and dark tobaccos. A common factor is that the curing is primarily free of artificial heat and moisture sources. Burley tobacco is light to dark brown in color, high in oil, and low in sugar. Burley tobacco is air-cured in sheds. The major growing countries for burley are Argentina, Brazil, Italy, Malawi, and the United States. Maryland tobacco is very fluffy, has good burning characteristics, is low in nicotine, and has a natural aroma. The major growing countries for Maryland include the United States and Italy. In one aspect, the provided low alkaloid or low nicotine tobacco plants or seeds are in a burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky14, Ky907, Ky910, Ky8959, NC2, NC3, NC4, NC5, NC2000, TN86, TN90, TN97, R610, R630, R711, R712, NCBH129, Bu21xKy10, HB04P, Ky14xL8, Kt200, Newton 98, Pedigo 561, Pf561, and Va509. In a further aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or strain is on any Burley background selected from the group consisting of TN90, KT209, KT206, KT212, and HB4488. In another aspect, the provided low alkaloid or low nicotine tobacco plant or seed is on a Maryland tobacco background selected from the group consisting of Md10, Md40, Md201, Md609, Md872, and Md341.

In one aspect, the tobacco plants provided are on a background of dark air-cured tobacco or exhibit one or more characteristics of dark air-cured tobacco described herein. Dark air-cured tobacco is primarily differentiated from other varieties by its curing process, which gives dark air-cured tobacco its medium to dark brown color and unique aroma. Dark air-cured tobacco is primarily used for chewing tobacco and snuff. In one aspect, the low alkaloid or low nicotine tobacco plants or seeds are provided on a background of dark air-cured tobacco selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado.

In one aspect, the provided tobacco plants are in the context of dark flame-cured tobacco or exhibit one or more characteristics of dark flame-cured tobacco described herein. Dark flame-cured tobacco is typically cured over low-burning open fires on the floor of an enclosed curing shed. Their leaves have a low sugar content but a high nicotine content. Dark flame-cured tobacco is used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong-flavored cigars. The primary growing areas for dark flame-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In one aspect, the low alkaloid or low nicotine tobacco plants or seeds provided are selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, and others. Mammoth), DT508, DT518, DT592, KY171, DF911, DF485, TND94, TND950, VA309, and VA359.

In one aspect, the provided tobacco plants are in the Oriental tobacco background or exhibit one or more Oriental tobacco characteristics described herein. Oriental tobacco is also referred to as Greek tobacco, aromatic tobacco, and Turkish tobacco due to the fact that it is typically grown in the Eastern Mediterranean region, such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant and leaf size characteristic of today's Oriental varieties, as well as their specific aromatic characteristics, are the result of the plant's adaptation to poor soil and stressful climatic conditions, developed over the past few centuries. In one aspect, the provided low alkaloid or low nicotine tobacco plants or seeds are from the following regions: Izmir, Katerini, Samsun, Basma, and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Şemdinli, Adyar, and others. In the background of the oriental tobacco is selected from the group consisting of Adiyanman, Yayladag, Iskenderun, Düzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir and any variety essentially derived from any one of the aforementioned varieties.

In one aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, cultivar, or strain is selected from the group consisting of BU64, CC101, CC200, CC27, CC301, CC400, CC500, CC600, CC700, CC800, CC900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU263, DF911, Galpao tobacco, GL26H, GL350, GL600, GL737, GL939, GL973, HB04P, K149, K326, K346, K358, K394, K399, K730, KDH959, KT200, KT204LC, KY10, KY14, KY160, KY17, KY171, KY907, KY907LC, KTY14xL8LC, Little Crittenden, McNair373, McNair944, msKY14xL8, Narrow Leaf Madol, NC100, NC102, NC2000, NC291, NC297, NC299, NC3, NC4, NC5, NC6, NC7, NC606, NC71, NC72, NC810, NCBH129, NC2002, Neal Smith Madol Madole), OXFORD 207, "Perique" tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG17, RG81, RGH51, RGH4, RGH51, RS1410, Speight168, Speight172, Spe light179, Speight210, Speight220, Speight225, Speight227, Speight234, SpeightG-28 , SpeightG-70, SpeightH-6, SpeightH20, SpeightNF3, TI1406, TI1269, TN86, TN86LC, TN9 0, TN97, TN97LC, TND94, TND950, TR (Tom Rosson) Madol, VA309, or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, Shirey LC, or any commercially available tobacco variety by standard tobacco breeding techniques known in the art, or in the genetic background thereof.

All specific varieties of the aforementioned dark air-cured, burley, Maryland, dark flame-cured, or Oriental varieties are listed for illustrative purposes only. Any additional dark air-cured, burley, Maryland, dark flame-cured, or Oriental varieties are also contemplated by this application.

Also provided are populations of the described tobacco plants. In one aspect, the population of tobacco plants has a planting density of about 5,000 to about 8000, about 5,000 to about 7,600, about 5,000 to about 7,200, about 5,000 to about 6,800, about 5,000 to about 6,400, about 5,000 to about 6,000, about 5,000 to about 5,600, about 5,000 to about 5,200, about 5,200 to about 8,000, about 5,600 to about 8,000, about 6,000 to about 8,000, about 6,400 to about 8,000, about 6,800 to about 8,000, about 7,200 to about 8,000, or about 7,600 to about 8,000 plants per acre. In another embodiment, the tobacco plant population is in a soil type having low to medium fertility.

Also provided is a container of seeds from the described tobacco plants. The container of tobacco seeds of the present disclosure may contain any number, weight, or volume of seeds. For example, the container can contain about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can contain about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds, or more seeds. The container of tobacco seeds may be any container available in the art. As non-limiting examples, the container may be a box, bag, packet, pouch, tape roll, tube, or bottle.

Cured tobacco materials made from the described low alkaloid or low nicotine tobacco plants are also provided. Additionally, cured tobacco materials made from the described tobacco plants with higher levels of total alkaloids or nicotine are provided.

"Curing" is a retrogradation process that reduces moisture and results in the breakdown of chlorophyll, giving tobacco leaves a golden color and converting starch to sugars. Thus, cured tobacco has a higher reducing sugar content and a lower starch content compared to harvested green leaves. In one aspect, the green-leaf tobacco provided can be dried using conventional means, such as hot air curing, barn drying, open flame drying, air drying, or sun drying. For a description of different types of curing methods, see, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is typically aged in compressed conditions for several years (e.g., 2-5 years) in wooden drums (e.g., hogsheads) or cardboard boxes, with moisture contents ranging from 10% to about 25%. See U.S. Patent Nos. 4,516,590 and 5,372,149. The dried and aged tobacco can then be further processed. Further processing includes the introduction of steam at various temperatures, pasteurization, and conditioning the tobacco under vacuum with or without fermentation. Fermentation is typically characterized by an initial high moisture content, the production of heat, and a 10-20% loss in dry weight. See, for example, U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149, U.S. Publication No. 2005/0178398, and Tso (1999, Chapter 1 of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The cured, aged and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Patent Nos. 4,528,993, 4,660,577, and 4,987,907. In one aspect, the dried tobacco material of the present disclosure is sun-dried. In another aspect, the dried tobacco material of the present disclosure is hot-air-dried, air-dried, or flame-dried.

The presence of mold on cured tobacco can significantly reduce the quality and marketability (e.g., leaf grade) of the cured leaf. Mold growth is a common problem that can occur at temperatures between about 10°C (50°F) and 32.2°C (90°F) and high humidity (e.g., greater than 70% relative humidity) for extended periods of time. Mold tends to be more prevalent at higher temperatures.

Tobacco plants, varieties, and strains provided herein that include mutant alleles in one or more PMT genes, two or more PMT genes, three or more PMT genes, four or more PMT genes, or five PMT genes exhibit reduced mold infection compared to the low-alkaloid tobacco cultivar LA Burley 21 (LA BU21). Similarly, tobacco plants, varieties, and strains provided herein that include RNAi constructs that downregulate the expression or translation of one or more PMT genes, two or more PMT genes, three or more PMT genes, four or more PMT genes, or five PMT genes exhibit reduced mold infection compared to the low-alkaloid tobacco cultivar LA Burley 21 (LA BU21).

LA BU21 is a low total alkaloid tobacco strain produced by incorporating low alkaloid gene(s) from Cuban Cigar varieties into Burley 21 through multiple backcrosses (Legg et al., Crop Science, 10:212 (1970)). It has about 0.2% total alkaloids (dry weight) compared to about 3.5% (dry weight) of its parent, Barley 21. LA BU21 has a leaf grade well below commercially acceptable standards.

In one aspect, cured tobacco leaves containing mutant alleles of pmt1a are free of observable mold infections. In another aspect, cured tobacco leaves containing mutant alleles of pmt1b are free of observable mold infections. In another aspect, cured tobacco leaves containing mutant alleles of pmt2 are free of observable mold infections. In another aspect, cured tobacco leaves containing mutant alleles of pmt3 are free of observable mold infections. In another aspect, cured tobacco leaves containing mutant alleles of pmt4 are free of observable mold infections. In another aspect, cured tobacco leaves containing mutant alleles of pmt1a, pmt1b, pmt2, pmt3, and pmt4 are free of observable mold infections.

In one aspect, cured tobacco leaves comprising mutant alleles of pmt1a comprise reduced mold infection compared to control cured tobacco leaves derived from various LA BU21. In another aspect, cured tobacco leaves comprising mutant alleles of pmt1b comprise reduced mold infection compared to control cured tobacco leaves derived from various LA BU21. In another aspect, cured tobacco leaves comprising mutant alleles of pmt2 comprise reduced mold infection compared to control cured tobacco leaves derived from various LA BU21. In another aspect, cured tobacco leaves comprising mutant alleles of pmt3 comprise reduced mold infection compared to control cured tobacco leaves derived from various LA BU21. In another aspect, cured tobacco leaves comprising mutant alleles of pmt4 comprise reduced mold infection compared to control cured tobacco leaves derived from various LA BU21. In another aspect, cured tobacco leaves containing mutant alleles of pmt1a, pmt1b, pmt2, pmt3, and pmt4 contain reduced mold infection compared to control cured tobacco leaves from cultivar LA BU21.

In one aspect, cured leaves from a tobacco plant, variety, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14 are free of observable mold infection. In another aspect, cured leaves from a tobacco plant, variety, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14 are free of observable mold infection compared to a control cured tobacco leaf from variety LA BU21.

In one aspect, cured leaves from a tobacco plant, variety, or strain containing one or more pmt mutations provided in any one of Tables 5A-5E and Tables 12A-12E are free of observable mold infection. In another aspect, cured leaves from a tobacco plant, variety, or strain containing one or more pmt mutations provided in any one of Tables 5A-5E and Tables 12A-12E contain reduced mold infection compared to control cured leaves from variety LA BU21.

In one aspect, cured leaves from a tobacco plant, variety, or strain containing a mutant allele of pmt1a comprise a higher leaf grade than a control cured leaf from variety LA BU21. In one aspect, cured leaves from a tobacco plant, variety, or strain containing a mutant allele of pmt1b comprise a higher leaf grade than a control cured leaf from variety LA BU21. In one aspect, cured leaves from a tobacco plant, variety, or strain containing a mutant allele of pmt2 comprise a higher leaf grade than a control cured leaf from variety LA BU21. In one aspect, cured leaves from a tobacco plant, variety, or strain containing a mutant allele of pmt3 comprise a higher leaf grade than a control cured leaf from variety LA BU21. In one aspect, cured leaves from a tobacco plant, variety, or strain containing a mutant allele of pmt4 comprise a higher leaf grade than a control cured leaf from variety LA BU21. In another aspect, cured tobacco leaves from a plant, cultivar, or strain containing a mutant allele of pmt1a, a mutant allele of pmt1b, a mutant allele of pmt2, a mutant allele of pmt3, and a mutant allele of pmt4 contain a higher leaf grade than a control cured leaf of LA BU21.

In one aspect, cured leaves from a tobacco plant, variety, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14 include a higher leaf grade than a control cured leaf from variety LA BU21.

In one aspect, cured leaves from a tobacco plant, cultivar, or line containing one or more pmt mutations provided in any one of Tables 5A-5E and Tables 12A-12E contain a higher leaf grade than a control cured leaf from cultivar LA BU21.

In one aspect, "reduced mold infection" refers to reduced infected leaf area. In another aspect, "reduced mold infection" refers to reduced number of surviving mold spores on infected leaves. Standard methods for detecting and counting viable spores are known and available in the art.

In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 1% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 2% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 3% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 4% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 5% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 10% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 15% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 20% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of at least 25% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 30% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 35% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 40% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 50% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 60% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 70% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 75% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 80% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area by at least 90% compared to control leaves. In one aspect, reduced mold infection includes a reduction in infected leaf area by at least 95% compared to control leaves. In one aspect, reduced mold infection includes a reduction in infected leaf area by 100% compared to control leaves.

In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 100% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 90% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 80% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 70% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 60% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 50% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 40% compared to control leaves. In one aspect, the reduced mold infection comprises a reduction in infected leaf area of 1% to 30% compared to control leaves. In one aspect, reduced mold infection comprises a 1% to 20% reduction in infected leaf area compared to control leaves. In one aspect, reduced mold infection comprises a 1% to 10% reduction in infected leaf area compared to control leaves. In one aspect, reduced mold infection comprises a 10% to 100% reduction in infected leaf area compared to control leaves. In one aspect, reduced mold infection comprises a 20% to 100% reduction in infected leaf area compared to control leaves. In one aspect, reduced mold infection comprises a 30% to 100% reduction in infected leaf area compared to control leaves. In one aspect, reduced mold infection comprises a 40% to 100% reduction in infected leaf area compared to control leaves. In one aspect, reduced mold infection comprises a 50% to 100% reduction in infected leaf area compared to control leaves. In one aspect, reduced mold infection comprises a 60% to 100% reduction in infected leaf area compared to control leaves. In one aspect, the reduced mold infection comprises a 70% to 100% reduction in infected leaf area compared to control leaves. In one aspect, the reduced mold infection comprises an 80% to 100% reduction in infected leaf area compared to control leaves. In one aspect, the reduced mold infection comprises a 90% to 100% reduction in infected leaf area compared to control leaves. In one aspect, the reduced mold infection comprises a 10% to 75% reduction in infected leaf area compared to control leaves. In one aspect, the reduced mold infection comprises a 25% to 75% reduction in infected leaf area compared to control leaves. In one aspect, the reduced mold infection comprises a 25% to 50% reduction in infected leaf area compared to control leaves.

In one embodiment, the mold infecting the cured tobacco is a mold of a genus selected from the group consisting of Cladosporium, Penicillium, Alternaria, Aspergillus, and Mucor.

Tobacco materials obtained from the tobacco strains, varieties, or hybrids of the present disclosure can be used to make tobacco products. As used herein, "tobacco product" is defined as any product made or derived from tobacco intended for human use or consumption.

Tobacco products provided include, but are not limited to, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigars and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, molded parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, and the like. See, e.g., U.S. Patent Publication No. US 2006/0191548.

As used herein, a "cigarette" refers to a tobacco product having a "rod" and a "filler." The "rod" of a cigarette includes the cigarette paper, the filter, the plug wrap (used to contain the filtration material), the tipping paper that holds the cigarette paper (including the filler) to the filter, and any adhesives that hold these components together. "Filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other spices that may accompany tobacco wrapped in cigarette paper), (3) casing, (4) flavorings, and (5) all other additives (mixed with the tobacco and substitutes and wrapped within the cigarette).

As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco scrap materials, processed into sheet form, and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is of great importance because it contributes to the taste of a cigarette through the process of flavor development using a reaction between ammonia and sugars.

As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been processed through expansion of a suitable gas so that the tobacco is "filled," reducing its density and increasing its fill capacity, thereby reducing the weight of the tobacco used in the cigarette.

The plant-derived tobacco products of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles that include a filter element, and the rod of smokable material includes dried tobacco in a tobacco blend. In one aspect, the tobacco product of the present disclosure is selected from the group consisting of cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, the tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products are not burned and include, but are not limited to, chewing tobacco, moist smokeless tobacco, snuff, and dry snuff. Chewing tobacco is coarsely divided tobacco leaves, typically packaged in large pouch-like packages and used for plugs and twists. Moist smokeless tobacco is moist, finely divided tobacco, provided in loose form or in pouches, typically packaged in round tins and used as pinches or pouches by adult tobacco consumers to be placed between the cheek and gums. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco that is placed in the mouth or used in the nose. In a further aspect, the tobacco product of the present disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of electronically heated cigarettes, electronic cigarettes, and electronic vaporization devices.

In one aspect, the tobacco product of the present disclosure may be a blended tobacco product. In another aspect, the tobacco product of the present disclosure may be a low-nicotine tobacco product. In a further aspect, the tobacco product of the present disclosure may include nornicotine at a level of less than about 3 mg/g. For example, the nornicotine content in such products may be 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 μg/g, 500 pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g, 5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g, 0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable.

In one aspect, the dried tobacco material or tobacco product provided has a dry weight basis of about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8% , 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9%. In another aspect, the provided dried tobacco material or tobacco product has about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, 1.4% to 1.5%, or %, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2%, 2% to 2.1%, 2.1% to 2.2%, 2.2% to 2.3%, 2.3% to 2.4%, 2.4% to 2.5%, 2.5% to 2.6%, 2.6% to 2.7%, 2.7% to 2.8%, 2.8% to 2.9%, 2.9% to 3%, 3% to 3.1%, 3.1% to 3.2%, 3.2% to 3.3%, 3.3% to 3.4%, 3.4% to 3.5%, and 3.5% to 3.6%. In a further aspect, the provided dried tobacco material or tobacco product comprises an average nicotine level or total alkaloid level selected from the group consisting of about 0.01% to 0.1%, 0.02% to 0.2%, 0.03% to 0.3%, 0.04% to 0.4%, 0.05% to 0.5%, 0.75% to 1%, 0.1% to 1.5%, 0.15% to 2%, 0.2% to 3%, and 0.3% to 3.5% on a dry weight basis.

The present disclosure also provides a method for breeding tobacco strains, cultivars, or varieties that contain a desired level of total alkaloids or nicotine, e.g., low nicotine or nicotine free. Breeding can be performed via any known procedure. DNA fingerprinting, SNP mapping, haplotype mapping, or similar techniques may be used in marker-assisted selection (MAS) breeding programs to transfer or breed desirable traits or alleles into tobacco plants. For example, breeders can use F1 hybrid plants to create segregating populations in the _F2 or backcross generation, or further cross F1 hybrid plants with other donor plants that have agronomically desirable genotypes. Plants in the _F2 or backcross generation can be screened for desirable agronomic traits or desirable chemical properties using one of the techniques known in the art or listed herein. Depending on the expected inheritance pattern or MAS technique used, self-pollination of selected plants can be performed before each cycle of backcrossing to aid in the identification of desirable individual plants. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is restored. The recurrent parent in this disclosure can be a flue-cured variety, a burley variety, a dark air-cured variety, a dark flame-cured variety, or an oriental variety. Other breeding techniques are described, for example, in Wernsman, E. A., and Rufty, R. C. 1987 Chapter Seventeen. Tobacco. Pages 669-698 Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. (which are incorporated herein by reference in their entireties).

The results of a plant breeding program using the described tobacco plants include useful strains, cultivars, varieties, progeny, inbreds, and hybrids of the present disclosure. As used herein, the term "variety" refers to a population of plants that share certain characteristics that separate them from other plants of the same species. Varieties are often, but not always, sold commercially. While a variety has one or more characteristic traits, it is further characterized by very little overall variation among individuals within the variety. "Pure line" varieties can be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. A variety can be essentially derived from another strain or variety. As defined by the International Convention for the Protection of New Varieties of Plants (December 2, 1961, November 10, 1972, October 23, 1978, and March 19, 1991, as amended at Geneva), a variety is "essentially derived" from an original variety if it a) is derived primarily from the original variety or is derived from a variety derived primarily from the original variety while retaining the expression of essential characteristics resulting from the genotype or combination of genotypes of the original variety, b) is clearly distinguishable from the original variety, and c) matches the original variety in the expression of essential characteristics resulting from the genotype or combination of genotypes of the original variety, except for differences resulting from acts of derivation. Essentially derived varieties can be obtained, for example, by selection of natural or induced mutants, somaclonal variants, variant individuals derived from plants of the original variety, backcrossing, or transformation. A first tobacco variety and a second tobacco variety from which the first variety is essentially derived are considered to have essentially the same genetic background. A "strain", as distinguished from a variety, most often refers to a group of plants used non-commercially, for example in botanical research. Strains typically show little overall variation among individuals for one or more traits of interest, although there may be some variation among individuals for other traits.

In one aspect, the present disclosure provides a tobacco plant, cultivar, strain, or cell that includes one or more pmt mutations provided in any one of Tables 5A-5E and Tables 12A-12E.

In another aspect, the present disclosure provides a tobacco plant, cultivar, strain, or cell derived from any tobacco plant, cultivar, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14.

In one aspect, the present disclosure provides tobacco line 18GH203, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH341, and F 1 or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1678, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1680, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1804, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived _therefrom . In one aspect, the present disclosure provides tobacco line 17GH1898, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH207, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH342, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH343, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH348, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH349, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH355, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH359, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH64, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH682, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH692, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH697, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH922, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH957, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1808, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1810, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1886, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1888, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1889, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH189, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1893, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1901, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1902, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH3, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH125, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH208, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH403, and F 1 or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH414, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH434, and F ₁ or F ₂ tobacco plants, or male sterile tobacco _{plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH436, and F 1 or F 2 tobacco plants} , or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH437, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH449, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH706, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH709, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH710, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH716, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH729, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH731, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH752, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH756, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH768, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH771, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH776, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH800, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH818, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides a tobacco line 18GH10, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides a tobacco line 18GH1004, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides a tobacco line 18GH1033, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides a tobacco line 18GH132, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides a tobacco line 18GH134, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH217, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH456, and F 1 or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH457, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH460, and F ₁ or F ₂ tobacco plants, or male sterile tobacco _{plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH465, and F 1 or F 2 tobacco plants} , or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH71, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH830, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH831, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH836, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH841, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH974, and _F1 or _F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH981, and F
In one aspect, the present disclosure provides a tobacco line 18GH994, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides a tobacco line 17GH1905, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides a tobacco line 18GH128, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived _{therefrom. In one aspect, the present disclosure provides a tobacco line 18GH130, and an F 1 or} F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides a tobacco line 18GH131, and _{an F 1 or} F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH133, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH136, and F 1 or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH216, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH227, and F ₁ or F ₂ tobacco plants, or male sterile tobacco _{plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH5, and F 1 or F 2 tobacco plants} , or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH6, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH65, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH66, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH69, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH72, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH73, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH74, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH78, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH79, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH8, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH9, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1696, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1717, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1719, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1729, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1736, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1737, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1739, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1740, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1835, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1848, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1849, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1912, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1937, and F 1 or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1940, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1943, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1944, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived _therefrom . In one aspect, the present disclosure provides tobacco line 18GH1051, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH22, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH34, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH473, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH49, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH50, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH848, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH850, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH851, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1699, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1708, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1722, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1724, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1725, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1845, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1846, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1847, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1911, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1912, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1915, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1918, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1928, and F 1 or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1932, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1933, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1936, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived _therefrom . In one aspect, the present disclosure provides tobacco line 18GH20, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH28, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH31, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH47, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH51, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH52, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS107, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS106, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS115, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1809-13, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS111, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS112, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1678-60, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS131, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH709-01, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH709-08, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH414-11, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH414-19, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH437-04, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH437-08, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH437-32, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH437-39, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH449-26, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH449-33, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH125-48, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides tobacco line CS102, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides tobacco line CS103, and an F ₁ or F ₂ tobacco plant, or a male sterile tobacco plant derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1719-30, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1740-36, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1698-22, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1700-13, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1702-17, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1849-01, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1849-48, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 17GH1737-24, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS118, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS133, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS120, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH1108-07, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH2162, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS164, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS163, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS146, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS147, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS150, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS151, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS148, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS149, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS152, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS153, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS143, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH2169, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH2171, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS165, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line CS118, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom. In one aspect, the present disclosure provides tobacco line 18GH2254-7, and F ₁ or F ₂ tobacco plants, or male sterile tobacco plants derived therefrom.

In one aspect, the disclosure provides a method of introgressing a low-nicotine trait into a tobacco variety, comprising: (a) crossing a first tobacco variety including the low-nicotine trait with a second tobacco variety not having the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for pmt mutant alleles selected from those listed in Tables 4A-4E, 5A-5E, 10, and 12A-12E; and (c) selecting a progeny tobacco plant including the pmt mutant allele. In another aspect, the methods further comprise backcrossing the selected progeny tobacco plant with the second tobacco variety. In a further aspect, the methods further comprise: (d) crossing the selected progeny plant with itself or the second tobacco variety to produce one or more additional progeny tobacco plants; and (e) selecting an additional progeny tobacco plant including the low-nicotine trait. In one aspect, the selecting of step (e) comprises marker-assisted selection. In one aspect, these methods produce single gene conversions comprising a low nicotine trait. In one aspect, these methods produce single gene conversions comprising pmt mutant alleles. In one aspect, the second tobacco variety is an elite variety. In another aspect, the genotyping step of these methods involves one or more molecular marker assays. In another aspect, the genotyping may comprise polymorphic markers comprising polymorphisms selected from the group consisting of single nucleotide polymorphisms (SNPs), insertions or deletions (indels) in DNA sequences, simple sequence repeats (SSRs) of DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs.

As used herein, a "locus" is a chromosomal locus or region at which a polymorphic nucleic acid, trait determinant, gene, or marker is located. A "locus" may refer to a corresponding locus or region shared by two homologous chromosomes. As used herein, an "allele" refers to an alternative nucleic acid sequence at a gene or a particular locus (e.g., a nucleic acid sequence at a gene or locus for the same gene or locus that is different from other alleles). Such an allele may be considered (i) wild type, or (ii) mutant if one or more mutations or edits are present in the nucleic acid sequence of the mutant allele compared to the wild type allele. A mutant allele of a gene may have a reduced or eliminated activity or expression level of the gene compared to the wild type allele. For a diploid organism such as tobacco, a first allele may occur on one chromosome and a second allele may occur at the same locus on a second homologous chromosome. If one allele at a locus on one chromosome of a plant is a mutant allele and the other corresponding allele on the plant's homologous chromosome is wild type, the plant is described as heterozygous for the mutant allele. However, if both alleles at a locus are mutant alleles, the plant is described as homozygous for the mutant allele. Plants that are homozygous for mutant alleles at a locus may contain the same mutant allele or different mutant alleles, if heteroallelic or biallelic.

As used herein, "transgenic" or "transgenic" refers to the transfer of a desired allele at one genetic locus to another locus from one genetic background.

As used herein, "crossed" or "crossing" means producing offspring through fertilization (e.g., of a cell, seed, or plant), and includes crossing between plants (sexual) and self-fertilization (self-pollination).

As used herein, "backcross" and "backcrossing" refer to the process of repeatedly crossing a progeny plant back to one of its parents. In a backcrossing scheme, the "donor" parent refers to the parent plant that has the desired gene or locus to be transgenic. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is transgenic. The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on. In one aspect, backcrossing is performed repeatedly, with each successive backcross generation of progeny individuals being themselves backcrossed to the same parental genotype.

As used herein, "single-gene transformed" or "single-gene transformation" refers to a plant that has been developed using a plant breeding technique known as backcrossing or through genetic engineering, and in which essentially all of the desired morphological and physiological characteristics of the variety are restored in addition to the transfer of a single gene into the variety through the backcrossing technique or through genetic engineering.

As used herein, "elite variety" means any variety that has resulted from breeding and selection for superior agronomic performance.

As used herein, "selecting" or "selection" in the context of marker-assisted selection or breeding refers to the act of picking or choosing desired individuals, usually from a population, based on certain predetermined criteria.

As used herein, the term "trait" refers to one or more detectable characteristics of a cell or organism that can be influenced by genotype. A phenotype may be observable with the naked eye or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, genomic analysis, assays for specific disease resistance, etc. In some cases, a phenotype is directly controlled by a single gene or locus, e.g., a "monogenic trait." In other cases, a phenotype is due to several genes.

As used herein, "marker assay" refers to a method that detects a polymorphism at a particular locus using a particular method, such as, for example, measurement of at least one phenotype (such as seed color, flower color, or other visually detectable trait), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), microarray-based techniques, and nucleic acid sequencing techniques.

As used herein, "marker assisted selection" (MAS) is a process in which a phenotype is selected based on a marker genotype. "Marker assisted selection breeding" refers to the process of selecting a desired trait(s) in a plant(s) by detecting one or more nucleic acids from the plant, linking those nucleic acids to the desired traits, and then selecting plants or germplasm that have those one or more nucleic acids.

As used herein, "polymorphism" refers to the presence of one or more variations in a population. Polymorphisms can manifest as variations in the nucleotide sequence of a nucleic acid or as variations in the amino acid sequence of a protein. Polymorphisms include the presence of one or more variations in a nucleic acid sequence or nucleic acid feature at one or more loci in a population of one or more individuals. Variations can include, but are not limited to, base changes of one or more nucleotides, insertions of one or more nucleotides, or deletions of one or more nucleotides. Polymorphisms can arise from random processes in nucleic acid replication, through mutagenesis, as a result of mobile genomic elements, from copy number variations, and during the process of meiosis (e.g., unequal crossing over, genome duplication, and chromosome breakage and chromosome fusion). Variations can be commonly found or present at low frequencies within a population, with the former having greater utility in general plant breeding and the latter being associated with rare but important phenotypic variations. Useful polymorphisms can include single nucleotide polymorphisms (SNPs), insertions or deletions in DNA sequences (Indels), simple sequence repeats (SSRs) in DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs. Also, genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, 5' untranslated regions of genes, 3' untranslated regions of genes, microRNAs, siRNAs, resistance loci, satellite markers, transgenes, mRNAs, dsmRNAs, transcriptional profiles, and methylation patterns can include polymorphisms. In addition, the presence, absence, or variation of the copy numbers of the above can also include polymorphisms.

As used herein, "SNP" or "single nucleotide polymorphism" means a sequence variation that occurs when a single nucleotide (A, T, C, or G) in a genomic sequence is changed or variable. A "SNP marker" exists when a SNP is mapped to a site on the genome.

As used herein, the terms "marker" or "molecular marker" or "marker locus" are used to indicate a nucleic acid or amino acid sequence that is sufficiently unique to characterize a particular locus on the genome. Any detectable polymorphic trait can be used as a marker as long as it is differentially inherited and exhibits linkage disequilibrium with the phenotypic trait of interest. Thus, each marker is an index of a particular segment of DNA with a unique nucleotide sequence. The map position provides a measure of the relative location of particular markers with respect to each other. When a trait is described as being associated with a given marker, it will be understood that the actual DNA segment whose sequence affects the trait generally cosegregates with the marker. More precise and reliable localization of the trait can be obtained when markers are identified on both sides of the trait. Relatively simple molecular tests can detect the presence of a trait by measuring the appearance of a marker(s) in the progeny of a cross, without actually assessing the appearance of the trait itself (which can be difficult and time-consuming because it requires growing plants at a stage and/or under environmental conditions in which the trait can be expressed).

It is understood that any tobacco plant of the present disclosure may further include additional agronomically desirable traits, for example, by transformation with a genetic construct or transgene using techniques known in the art. Examples of desirable traits include, but are not limited to, herbicide resistance, pest resistance, disease resistance, high yield, high grade index value, desiccation, drying quality, machine harvestability, retention, leaf quality, height, plant maturity (e.g., early maturity, early to mid maturity, mid maturity, mid to late maturity, or late maturity), stem size (e.g., small, medium, or large stem), or number of leaves per plant (e.g., low (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or high (16-21 leaves)), or any combination. In one aspect, the disclosed low-nicotine or nicotine-free tobacco plants or seeds contain one or more transgenes expressing one or more insecticidal proteins, such as the crystal protein of Bacillus thuringiensis or a plant insecticidal protein from Bacillus cereus, such as VIP3 (see, e.g., Estruch et al. (1997) Nat. Biotechnol. 15:137). In another aspect, the tobacco plants further contain a transgenic trait that confers resistance to brown stem rot (U.S. Pat. No. 5,689,035) or resistance to cyst nematodes (U.S. Pat. No. 5,491,081).

The present disclosure also provides pmt mutant tobacco plants that contain altered nicotine or total alkaloid levels, but have yields equivalent to the yields of the corresponding initial tobacco plants without such altered nicotine levels. In one aspect, the pmt mutants provide yields selected from the group consisting of about 1200-3500, 1300-3400, 1400-3300, 1500-3200, 1600-3100, 1700-3000, 1800-2900, 1900-2800, 2000-2700, 2100-2600, 2200-2500, and 2300-2400 pounds per acre. In another aspect, the pmt mutant tobacco variety provides a yield selected from the group consisting of about 1200-3500, 1300-3500, 1400-3500, 1500-3500, 1600-3500, 1700-3500, 1800-3500, 1900-3500, 2000-3500, 2100-3500, 2200-3500, 2300-3500, 2400-3500, 2500-3500, 2600-3500, 2700-3500, 2800-3500, 2900-3500, 3000-3500, and 3100-3500 pounds per acre. In further aspects, pmt mutant tobacco plants provide a yield of 65%-130%, 70%-130%, 75%-130%, 80%-130%, 85%-130%, 90%-130%, 95%-130%, 100%-130%, 105%-130%, 110%-130%, 115%-130%, or 120%-130% of the yield of a control plant having essentially the same genetic background, except for the pmt mutation(s). In further aspects, pmt mutant tobacco plants provide a yield of 70%-125%, 75%-120%, 80%-115%, 85%-110%, or 90%-100% of the yield of a control plant having essentially the same genetic background, except for the pmt mutation.

In one aspect, the disclosed tobacco plants (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco varieties) include modifications that impart desirable traits (e.g., low-nicotine, nicotine-free, or low-alkaloid) without substantially affecting traits selected from the group consisting of yield, maturity and senescence, susceptibility to insect feeding damage, polyamine content after pruning, chlorophyll levels, mesophyll cell number per unit leaf area, and end-product quality after curing.

In one aspect, the disclosed tobacco plants include modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further include traits substantially equivalent to an unmodified control plant, the traits being selected from the group consisting of yield, maturity and senescence, susceptibility to insect feeding damage, polyamine content after pruning, chlorophyll levels, mesophyll cell number per unit leaf area, and end product quality after drying.

In one aspect, the disclosed tobacco plants include modifications that confer a desired trait (e.g., low nicotine, nicotine free, or low alkaloids) and further include a yield that is greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135%, or greater than 140% relative to the yield of an unmodified control plant. In one aspect, the disclosed tobacco plants include modifications that confer a desired trait (e.g., low nicotine, nicotine free, or low alkaloids) and further include a yield that is greater than 70%-140%, 75%-135%, 80%-130%, 85%-125%, 90%-120%, 95%-115%, or 100%-110% relative to the yield of an unmodified control plant. In one aspect, the disclosed tobacco plants include modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further include a yield that is 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-100%, 95%-105%, 105%-115%, 110%-120%, 115%-125%, 120%-130%, 125-135%, or 130%-140% of the yield of an unmodified control plant.

In one aspect, the disclosed low-nicotine or nicotine-free tobacco varieties are adapted for mechanical harvesting. In another aspect, the disclosed low-nicotine or nicotine-free tobacco varieties are mechanically harvested.

In one aspect, the tobacco plant provided is a hybrid plant. The hybrid can be produced by preventing self-pollination of a female parent plant (e.g., a seed parent) of a first variety, fertilizing the female parent plant with pollen from a male parent plant of a second variety, and forming F1 hybrid seeds in the female plant. Self-pollination of the female plant can be prevented by emasculating the flower at an early stage of flower development. Alternatively, a form of male sterility can be used to prevent pollen formation in the female parent plant. For example, male sterility can be produced by male sterility (MS) or transgenic male sterility, where a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility. A female parent plant containing MS is particularly useful. In one aspect where the female parent plant is MS, pollen is collected from a male fertile plant and manually applied to the stigma of an MS female parent plant, and the resulting F1 seeds are harvested.

The plants can be used to form single-cross tobacco F1 hybrids. Pollen from a male parent plant is manually transferred to an emasculated or male-sterile female parent plant to form F1 seeds. Alternatively, a three-way cross can be performed in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, the F1 progeny of two different single crosses can be crossed with themselves to create a double-cross hybrid. When forming double-cross hybrids, the use of self-incompatibility can be particularly advantageous, preventing self-pollination of the female parent.

In one aspect, the low-nicotine or nicotine-free tobacco variety is male sterile. In another aspect, the low-nicotine or nicotine-free tobacco variety is cytoplasmic male sterile. Male-sterile tobacco plants may be produced by any method known in the art. Methods for producing male-sterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987 Chapter Seventeen. Tobacco. Pages 669-698 Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y., and in The Journal of Tobacco 2000, 144: 1111-1115, 1987, pp. 1111-1115, 1987. It is described on page 761.

In one aspect, the present disclosure provides a male sterile tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of Tables 5A-5E and Tables 12A-12E.

In another aspect, the present disclosure provides a male sterile tobacco plant, variety, or line derived from any tobacco plant, variety, or line provided in any one of Tables 4A-4E, Table 10, or Table 14.

In one aspect, the disclosure provides a male sterile line dCS11. In another aspect, the disclosure provides a male sterile line dCS12. In another aspect, the disclosure provides a male sterile line dCS13. In another aspect, the disclosure provides a male sterile line dCS14. In another aspect, the disclosure provides a male sterile line dCS15. In another aspect, the disclosure provides a male sterile line dCS16. In another aspect, the disclosure provides a male sterile line dCS17. In another aspect, the disclosure provides a male sterile line dCS18. In another aspect, the disclosure provides a male sterile line dS697.

In a further aspect, the tobacco parts provided include, but are not limited to, leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, sheaths, embryos, endosperms, explants, callus, tissue cultures, shoots, cells, and protoplasts. In one aspect, the tobacco parts provided do not include seeds. In one aspect, the present disclosure provides organs that are not tobacco plant cells, tissues, and reproductive material and do not mediate natural plant reproduction. In another aspect, the present disclosure also provides organs that are tobacco plant cells, tissues, and reproductive material and mediate natural plant reproduction. In another aspect, the present disclosure provides tobacco plant cells, tissues, and organs that cannot sustain themselves via photosynthesis. In another aspect, the present disclosure provides tobacco plant cells that are somatic. Somatic cells, as opposed to germ line cells, do not mediate plant reproduction.

The cells, tissues, and organs may be derived from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, sheaths, flowers, inflorescences, stalks, pedicels, styles, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem, and vascular tissues. In another aspect, the present disclosure provides a chloroplast of a tobacco plant. In a further aspect, the present disclosure provides an epidermal cell, a stomatal cell, a leaf or root hair, a storage root, or a tuber. In another aspect, the present disclosure provides a protoplast of a tobacco plant.

Those skilled in the art understand that tobacco plants reproduce naturally through seeds, rather than through asexual or vegetative propagation. In one aspect, the present disclosure provides tobacco endosperm. In another aspect, the present disclosure provides tobacco endosperm cells. In a further aspect, the present disclosure provides male or female sterile tobacco plants that are unable to reproduce without human intervention.

In one aspect, the disclosure provides a nucleic acid molecule comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-10, and fragments thereof. In one aspect, the disclosure provides a polypeptide or protein comprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15.

As used herein, the term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the same residues in the two sequences when aligned for maximum correspondence over a specified comparison window. When percentages of sequence identity are used with respect to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acid residues are replaced with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), thus not altering the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

The present disclosure further provides a method of producing a tobacco product comprising tobacco material from the disclosed tobacco plant. In one aspect, the method includes conditioning aged tobacco material produced from the tobacco plant to increase its moisture content from about 12.5% to about 13.5% to about 21% and blending the conditioned tobacco material to produce a desired blend. In one aspect, the method of producing a tobacco product further includes casing or flavoring the blend. Generally, during the casing process, casing or source materials are added to the blend to balance the chemical composition and thereby enhance their quality and develop certain desired flavor characteristics. Further details of the casing process can be found in Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999.

The provided tobacco material can also be processed using methods including, but not limited to, heat treatment (e.g., cooking, toasting), flavoring, enzymatic treatment, expansion, and/or drying. Both fermented and non-fermented tobaccos can be processed using these techniques. Examples of suitable processed tobaccos include dark air-cured, dark flame-cured, burley, hot-air-cured, and cigar filler or wrapper, as well as products from whole leaf stemming operations. In one aspect, the tobacco fiber comprises up to 70% dark tobacco on a green weight basis. For example, the tobacco can be conditioned by heating, sweating, and/or pasteurization steps as described in U.S. Publication Nos. 2004/0118422 or 2005/0178398.

The provided tobacco material can be fermented. Fermentation is typically characterized by an initial high moisture content, heat generation, and a 10-20% loss in dry weight. See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, and 5,372,149. Fermentation can modify the aroma of the leaves, as well as change either or both of the color and texture of the leaves. Also, during the fermentation process, gases can be released, oxygen can be taken up, the pH can be changed, and the amount of moisture retained can be altered. See, for example, U.S. Publication No. 2005/0178398 and Tso (1999, Chapter 1 of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried or dried and fermented tobacco can be further processed (e.g., cut, expanded, blended, milled, or ground) before being incorporated into an oral product. The tobacco is optionally long-cut, fermented, dried, moist tobacco having an oven volatiles content of 48-50% by weight before being mixed with the copolymer and, optionally, flavorants and other additives.

In one aspect, the provided tobacco material may be processed to a desired size. In one aspect, the tobacco fibers may be processed to have an average fiber size of less than 200 micrometers. In one aspect, the tobacco fibers are 75-125 micrometers. In another aspect, the tobacco fibers are processed to have a size of 75 micrometers or less. In one aspect, the tobacco fibers include long cut tobacco that may be cut or chopped to a width of about 10 cuts/inch to about 110 cuts/inch and a length of about 0.1 inch to about 1 inch. The double cut tobacco fibers may have a range of particle sizes such that about 70% of the double cut tobacco fibers are of a mesh size of -20 mesh to 80 mesh.

The tobacco material provided can be processed to have a total oven volatiles content of about 10% by weight or more, about 20% by weight or more, about 40% by weight or more, about 15% to about 25% by weight, about 20% to about 30% by weight, about 30% to about 50% by weight, about 45% to about 65% by weight, or about 50% to about 60% by weight. Those skilled in the art will appreciate that "moist" tobacco typically refers to tobacco having an oven volatiles content of about 40% to about 60% by weight (e.g., about 45% to about 55% by weight, or about 50% by weight). As used herein, "oven volatiles" is determined by calculating the percentage of weight loss of a sample after drying the sample in a pre-warmed forced draft oven at 110° C. for 3.25 hours. Oral products can have a total oven volatiles content that is different from the oven volatiles content of the tobacco fiber used to make the oral product. The processing steps described can reduce or increase oven volatile content.

Having thus generally described the present disclosure, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the present disclosure, unless otherwise specified.

Example 1: Expression profiling of five PMT genes Nicotine biosynthesis begins with the conversion of the polyamine putrescine to N-methylputrescine by the enzyme putrescine N-methyltransferase (PMT), a step in which the precursor metabolite is involved in nicotine biosynthesis. Genes encoding PMTs (PMT1a, PMT1b, PMT2, PMT3, and PMT4) are present in the tobacco (Nicotiana tabacum) genome. Table 1A lists the genomic DNA, cDNA, and protein sequences of the five PMT genes. Tables 1B and 1C provide sequence identity among the five PMT genes. Pooled expression levels from pre-flowering to harvest provide support, without being limited to any particular theory, that PMT1a and PMT3 represent the two major PMT genes (Figure 1).

(Table 1A) Sequences of five tobacco PMT genes

(Table 1B) cDNA sequence identity among five tobacco PMT genes determined by Clustal2.1

(Table 1C) Protein sequence identity among the five tobacco PMT genes determined by Clustal2.1.

Table 1D. Annotation of PMT1b genomic sequence (SEQ ID NO:1).

Table 1E. Annotation of PMT1b genomic sequence (SEQ ID NO:2).

Table 1F. Annotation of PMT2 genomic sequence (SEQ ID NO:3)

Table 1G. Annotation of PMT3 genomic sequence (SEQ ID NO:4)

Table 1H. Annotation of PMT4 genomic sequence (SEQ ID NO:5)

Example 2: PMT genome editing and tobacco strain development PMT knockout mutants are generated by editing various PMT genes. Tobacco protoplasts are transfected with a plasmid encoding genome editing technology 1 (GET1) protein or genome editing technology (GET) 2 protein, and a specific guide RNA (gRNA) that targets the PMT gene at the desired position using polyethylene glycol (PEG). Table 2 lists the gRNA sequences used for PMT editing. Some gRNAs (e.g., numbers 6 and 7) are pooled together to target multiple PMT genes in a single transfection.

The transfected protoplasts are then fixed on 1% agarose beads and subjected to tissue culture. Once the calli have grown to approximately 1 mm in diameter, they are plated on TOM2 plates. The calli are screened for insertions or deletions (indels) at the target position using fragment analysis. Candidates that show a size shift compared to the wild-type control are selected for further culture, and the resulting shoots are again tested by fragment analysis to confirm the presence of indels. Rooted shoots are potted and sequenced at the target position to determine the exact deleted sequence. Young leaves from each plant are harvested and PCR amplified for the PMT fragment using phirekit. The PMT library is indexed for each line, and 384 lines are pooled and sequenced using Miseq.

SNP analysis is performed to determine both the sequence of the precise edited pmt mutant allele and the zygosity status at each PMT locus. Table 3 provides zygosity information for representative edited plants. Tables 4A-4E provide sequence information for indels in each edited line of various tobacco varieties (e.g., K326, TN90, NLM, Oriental). Tables 5A-5E provide genomic sequence of approximately 40 nucleotides from each pmt mutant allele with the edited site in the center of the genomic sequence (e.g., 20 nucleotides on each side of the sequence site of the deletion or insertion).

(Table 2) gRNA sequences and their target genes used in the two genome editing techniques. "Y" indicates that the gRNA targets that PMT gene, while "-" indicates that the gRNA does not target that PMT gene.

Table 3: Zygosity of individual PMT loci in selected pmt mutants in various backgrounds generated by genome editing using GET2. Number 1 represents homozygosity for a single mutant allele. Numbers 2-5 represent heteroallelic combinations with 2-5 indels. Hyphens indicate no data. Detailed genotype information is shown in Tables 4A-4D.

(Table 4A) Mutant pmt alleles in K326 generated by genome editing using GET2. The location of each edited site (e.g., indel) is relative to the nucleotide number on the corresponding cDNA sequence of each PMT gene. For example, strain 17GH1678 has a biallelic mutation in PMT1b. One of the two alleles has a four nucleotide deletion corresponding to nucleotides 416-419 of the PMT1b cDNA sequence. The other allele has a two nucleotide deletion corresponding to nucleotides 418-419 of the PMT1b cDNA sequence. For sequences of more than 10 nucleotides, SEQ ID NOs have been assigned and are shown.

(Table 4B) Mutant pmt alleles of TN90 generated by genome editing using GET2

(Table 4C) Mutant pmt alleles of NLMz generated by genome editing using GET2. NLMz refers to a narrow-leaf Madol cultivar that contains three loss-of-function mutations in three nicotine demethylase genes (CYP82E4, CYP82E5v2, and CYP82E10).

(Table 4D) Oriental tobacco mutant pmt alleles generated by genome editing using GET2

(Table 4E) Mutant pmt alleles of NLM (Ph Ph) tobacco generated by genome editing using GET1

(Table 5A) List of exemplary mutant alleles obtained in the PMT1b gene. The sequences of the mutant alleles listed herein and in Tables 5B-5E represent approximately 40 nucleotides in length of genomic sequence from each edited PMT gene with the edited site in the center of the genomic sequence (e.g., 20 nucleotides on each side of the sequence site of the deletion or insertion). These mutant alleles correspond to those listed in Tables 4A-4E.

(Table 5B) List of exemplary mutant alleles obtained in the PMT1a gene

(Table 5C) List of exemplary mutant alleles obtained in the PMT2 gene

Table 5D. List of exemplary mutant alleles obtained in the PMT3 gene

Table 5E: List of exemplary mutant alleles obtained in the PMT4 gene.

Example 3: Alkaloid Analysis of PMT-Edited Lines Genome-edited tobacco plants along with controls are grown in 10-inch pots in a greenhouse with 75 PPM fertilizer. At the flowering stage, plants are deflowered and two weeks after deflowering, leaf blade samples from the top third, fourth, and fifth leaves are collected and alkaloid levels are measured (see Tables 6A-6C). The measurements are performed using methods according to CORESTA Method No. 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatography Analysis (February 2005), and methods defined in the Centers for Disease Control and Prevention Protocol for Analysis of Nicotine, Total Moisture, and pH in Smokeless Tobacco Products, published in Federal Register Vol. 64, No. 55, March 23, 1999 (amended Vol. 74, No. 4, January 7, 2009).

Briefly, approximately 0.5 g of tobacco is extracted using liquid/liquid extraction into an organic solvent containing an internal standard and analyzed by gas chromatography (GC) with flame ionization detection (FID). Results can be reported as weight percent (wt%) either as is or on a dry weight basis. To report data on a dry weight basis, an oven volatiles (OV) determination is required. Unless otherwise stated, total or individual alkaloid or nicotine levels presented herein are on a dry weight basis (e.g., percent total alkaloids or percent nicotine).

Plants are also planted in the field, harvested, and tested for alkaloid and TSNA levels in the cured tobacco. Both leaf yield and leaf grade are also evaluated for the PMT edited plants. In addition, different mutant combinations of the individual PMT genes are generated and tested (e.g., single, double, triple, or quadruple mutants).

Example 4: Comparing the quintuple pmt knockout mutant to other low-alkaloid tobacco plants The quintuple pmt knockout mutant line CS15 (NLM(Ph Ph) background, see Table 4E for genotype) is grown side-by-side with a PMT RNAi transgene line (VA359 background, described in US 2015/0322451), and a low-nicotine KY171 ("LN KY171") cultivar (KY171 background with nic1 and nic2 double mutations). Leaves are harvested and dried via dark flame drying. Each line is analyzed for nicotine and total alkaloid levels, leaf yield, and leaf quality (Figures 2-5). The data show that suppressing the activity of the PMT genes by editing all five PMT genes reduces nicotine levels without affecting leaf yield or leaf quality.

Example 5: Obtaining tobacco lines with edited mutant alleles in one or more PMT genes Tobacco lines with mutations in individual PMT genes or selected combinations of PMT genes are obtained from the tobacco lines listed in Table 3. Quintuple, quadruple, triple, or double mutants (having mutations in five, four, three, or two PMT genes, respectively) are crossed with a non-mutated control line and segregating progeny plants are selected for the particular combination of PMT mutations. Tables 7A-7E represent possible mutant combinations that can be obtained. Each mutant gene can be either homozygous or heterozygous for the mutation. Each of the mutant alleles listed in Tables 4A-4E and 10 can be used to generate single, double, triple, quintuple, or quadruple mutants. Exemplary individual pmt mutant alleles are listed in Tables 9A-9E.

Example 6: Further reduction of total alkaloids by combining pmt mutation with mutations in other genes To further reduce total alkaloids and/or selected individual alkaloids, pmt mutants are combined with additional genes related to alkaloid biosynthesis in tobacco, such as quinolate phosphoribosyltransferase (QPT) or quinolinate synthase (QS) mutations. Briefly, gene editing is used to mutate selected QPT and/or QS genes in the desired pmt mutant background (e.g., quadruple or quinuple pmt mutants). The resulting combination of qpt/pmt or qs/pmt mutants is tested for alkaloid and TSNA levels in cured tobacco. Both leaf yield and leaf grade are also evaluated.

Table 6A. Alkaloid levels in PMT-edited lines of K326 (shown here and in Tables 6B, 6C, and 7 as percentage of weight per gram of leaf blade (dry weight)).

Table 6B. Alkaloid levels in PMT-edited strains of TN90

Table 6C. Alkaloid levels in PMT-edited strains of narrow-leaf madol (NLM).

Table 7: Relative changes in nicotine and total alkaloid levels in quintuple pmt knockout mutants in various cultivars. The average percent levels of nicotine and total alkaloids are calculated based on the percent level data from individual strains, as shown in Tables 6A-C. The relative changes reflect the nicotine or total alkaloid levels in quintuple pmt mutants compared to their controls.

(Table 8A) List of mutations obtained with various genotype combinations of the five PMT genes: single gene mutations

(Table 8B) List of mutants obtained with various genotype combinations of the five PMT genes: double gene mutations

(Table 8C) List of mutants obtained with various genotypic combinations of the five PMT genes: Triple gene combinations

(Table 8D) List of mutants obtained with various genotypic combinations of the five PMT genes: quadruple gene combinations

(Table 8E) List of mutants obtained with various genotypic combinations of the five PMT genes: quintuple gene combinations

Example 7: PMT genome editing and tobacco strain development Further PMT knockout mutants are generated by editing all five PMT genes (PMT1a, PMT1b, PMT2, PMT3, and PMT4) in different tobacco strains. Tobacco protoplasts are transfected with a plasmid encoding genome editing technology (GET2) protein and a specific guide RNA (gRNA) that targets the PMT gene at the desired position using polyethylene glycol (PEG). Table 9 lists the gRNA sequences used for PMT editing. Some gRNAs (e.g., numbers 6 and 7) are pooled together to target multiple PMT genes in a single transfection.

Table 9: Guide RNAs for GET2 used in Example 7. "Y" indicates that the gRNA can target that PMT gene, while "-" indicates that the gRNA does not target that PMT gene.

The transfected protoplasts are then fixed on 1% agarose beads and subjected to tissue culture. Once the calli have grown to approximately 1 mm in diameter, they are plated on TOM2 plates. The calli are screened for insertions or deletions (indels) at the target position using fragment analysis. Candidates that show a size shift compared to the wild-type control are selected for further culture, and the resulting shoots are again tested by fragment analysis to confirm the presence of indels. Rooted shoots are potted and sequenced at the target position to determine the exact deleted sequence. Young leaves from each plant are harvested and PCR amplified for the PMT fragment using phirekit. The PMT library is indexed for each line, and 384 lines are pooled and sequenced using Miseq.

SNP analysis is performed to determine both the exact edited pmt mutant allele sequences and the zygosity status at each PMT locus. Table 10 provides sequence information of indels in each edited line of various tobacco varieties (e.g., Basma, K326, Katerini, TN90, Izmir).

Table 10: Mutant pmt alleles of various strains generated by genome editing using GET2. The position of each edited site (e.g., indel) is relative to the nucleotide number on the corresponding cDNA sequence of each PMT gene (e.g., SEQ ID NO: 6 for PMT1a, SEQ ID NO: 7 for PMT1b, SEQ ID NO: 8 for PMT2, SEQ ID NO: 9 for PMT3, SEQ ID NO: 10 for PMT4). For sequences of more than 10 nucleotides, the sequence number is assigned and shown.

Table 11 provides the length (in nucleotides) of each PMT indel for each gene in each strain as provided in Table 10.

Table 11: Length (in nucleotides) of each indel in the selected strains provided in Table 10.

Tables 12A-12E provide approximately 90 nucleotides of genomic sequence from each pmt mutant allele with the editing site in the center of the genomic sequence (e.g., 45 nucleotides on each side of the sequence site of the deletion or insertion).

(Table 12A) List of exemplary mutant alleles obtained in the PMT1a gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides of genomic sequence from each edited PMT1a gene with the edited site in the center of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. The lowercase letters in the reference allele sequence (SEQ ID NO:6) indicate the nucleotides deleted in the mutant allele.

(Table 12B) List of exemplary mutant alleles obtained in the PMT1b gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides of genomic sequence from each edited PMT1b gene with the edited site in the center of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. The lowercase letters in the reference allele sequence (SEQ ID NO: 7) indicate the nucleotides deleted in the mutant allele.

(Table 12C) List of exemplary mutant alleles obtained in the PMT2 gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides in length of genomic sequence from each edited PMT2 gene with the edited site in the center of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. The lowercase letters in the reference allele sequence (SEQ ID NO: 8) indicate the nucleotides deleted in the mutant allele.

(Table 12D) List of exemplary mutant alleles obtained in the PMT3 gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides in length of genomic sequence from each edited PMT3 gene with the edited site in the center of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. The lowercase letters in the reference allele sequence (SEQ ID NO: 9) indicate the nucleotides deleted in the mutant allele.

(Table 12E) List of exemplary mutant alleles obtained in the PMT4 gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides in length of genomic sequence from each edited PMT4 gene with the edited site in the center of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. The lowercase letters in the sequence of the reference allele (SEQ ID NO: 10) indicate the nucleotides deleted in the mutant allele.

Example 8. Alkaloid Analysis of PMT-Edited Lines The homozygous genome-edited tobacco lines from Example 7, as well as the control lines, are grown in the field. At the flowering stage, the plants are deflowered, and two weeks after deflowering, leaf blade samples from the third, fourth, and fifth leaves from the top of the plants are collected and alkaloid levels are measured (see Tables 13A-13C). The measurements are performed using methods according to CORESTA Method No. 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatography Analysis (February 2005), and methods defined in the Centers for Disease Control and Prevention Protocol for Analysis of Nicotine, Total Moisture, and pH in Smokeless Tobacco Products, published in Federal Register Vol. 64, No. 55, March 23, 1999 (amended Vol. 74, No. 4, January 7, 2009).

Approximately 0.5 g of tobacco is extracted using liquid/liquid extraction into an organic solvent containing an internal standard and analyzed by gas chromatography (GC) with flame ionization detection (FID). Results can be reported as weight percent (wt%) either as is or on a dry weight basis. To report data on a dry weight basis, an oven volatiles (OV) determination is required. Unless otherwise stated, total or individual alkaloid or nicotine levels presented herein are on a dry weight basis (e.g., percent total alkaloids or percent nicotine).

Plants will also be planted in the field, harvested, and tested for alkaloid and TSNA levels in the cured tobacco. Both leaf yield and leaf grade will also be evaluated for the PMT-edited plants.

Table 13A. Nicotine analysis of K326 and TN90 PMT edited lines at 2 weeks after flowering

(Table 13B) Nicotine analysis of K326 and TN90 PMT edited lines 2 weeks after pruning

(Table 13C) Nicotine analysis of Katerini and Basma PMT edited lines at 2 weeks of flowering

Example 9. Development of male sterile PMT-edited lines PMT-edited hybrid lines are developed using lines from Example 7. The hybrid lines are grown in the field and used as precursors for male sterile lines. See Table 14.

Table 14. PMT-edited very low nicotine male sterile lines

Example 10. PMT edited strains are resistant to mold during drying Tobacco leaves from several low alkaloid tobacco strains are subjected to standard air-curing methods. After drying is completed, the tobacco leaves are inspected for mold.

Tobacco from LA BU21 shows more mold infection than TN90 LC, TN90 cultivars containing RNAi constructs downregulating all five PMT genes, TN90 cultivars containing RNAi constructs downregulating the alkaloid biosynthetic gene PR50, and four PMT edited lines (CS47, CS59, CS63, and CS64) in the TN90 genetic background. See Table 15 and Figures 6A-6E and 7.

Table 15: Mold damage by tobacco strain. "G" refers to little/no mold, "S" refers to some mold, and "B" refers to significant mold. The mold percentage refers to the proportion of air-dried sticks of tobacco that exhibit each category of mold damage.

Sequence information
SEQUENCE LISTING
<110> ALTRIA CLIENT SERVICES LLC
<120> COMPOSITIONS AND METHODS BASED ON PMT ENGINEERING FOR PRODUCING
TOBACCO PLANTS AND PRODUCTS HAVING ALTERED ALKALOID LEVELS
<150> US 62/703,775
<151> 2018-07-26
<150> US 62/848,159
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<213> Nicotiana tabacum
<400> 2
gaattttgta aatgcttttt ttggctcagt gattttgtgg agttgggaaa aatcctattg 60
gaaagtaggt cgtggttttt tcaccttttg aaccaggtat ttttcatgta aaaatacttg 120
tgttctttac tttttgcatt tactattcca caactgtagt gtaaggaaca cgtagaagaa 180
ccatgtccta taatctgtgc acgcgaaaaa ttggacacca cgcaaatcac ccttatgtg 240
tgtcattgaa atataaaaca tcaagctcaa tcatagatta atcttttttt agtaccaaca 300
tcatatgcaa aaatcaattc aaccccccaaa acataataca accaatgtta atgcaatatc 360
tctgctgcta tcacgaaaat aattgtagct cacgaaagta ggatacatta tgtaggttac 420
atcacataga ggtaatctaa agctcccaat aataagatgt gtaatgttga ttatgtagaa 480
atttgccagg ttatttagaa taaacaagaa gaggagaaaa aaagtacaat ttacctgaac 540
tcttgaatgt atcctacaaa taacctagac ttcatggacg tcagttgtca gtttactttt 600
gttttaatgg tacatcattt gtcaaatact ttattggat aaaaacagtt ttgcctaagg 660
agtaaacaga tccggagtaa gaaagcagac gattaaagca atttttaaaa aaggagagag 720
aaattaatga gcacacacat atactagtga aattagggta ctaatttact aataattgca 780
ccgagacaaa cttatatttt agttccaaaa tgtcagtcta accctgcacg ttgtaataaa 840
tttttaactc tattatatta tatcgagttg cgccctccac tcctcggtgt ccaaattgta 900
tttaaatgca tagatgttta atgggagtgt acagcaagct ttcggaaaat acaaaccata 960
atactttctc ttcttcaatt tgtttagttt aattttgaaa atggaagtca tatctaccaa 1020
cacaaatggc tctaccatct tcaagaatgg tgccattccc atgaacggcc accaaaatgg 1080
cacttctgaa cacctcaacg gctacgaa tggcacttcc aaacaccaaa acgggcacca 1140
gaatggcact ttcgaacatc ggaacggcca ccagaatggg acatccgaac aacagaacgg 1200
gacaatcagc catgacaatg gcaacgagct actgggaagc tccgactcta ttaagcctgg 1260
ctggttttca gagtttagcg cattatggcc aggttagtat taagaaagaa actcaaatgc 1320
atcgtactct tgtattttgg tttgtgtata atttataata tggataaatt atgacgaagt 1380
aaactcgcat acattaattt gaaataatct aattttgcag gtgaagcatt ctcacttaag 1440
gttgagaagt tactattcca ggggaagtct gattaccaag atgtcatgct ctttgaggta 1500
attaatattc tgatacacat gctttaatct aaagtgatac ttttaattta cttttagttt 1560
attgcatgtg cacgtacagt cagcaactta tgggaaggtt ctgactttgg atggagcaat 1620
tcaacataca gagaatggtg gatttccata cactgaaatg attgttcatc taccacttgg 1680
ttccatccca aaccccaaaaa aggttttgat catcggcgga ggaattggtt ttacattatt 1740
cgaaatgctt cgttatcctt caatcgaaaa aattgacatt gttgagatcg atgacgtggt 1800
agttgatgta agtcaaactt cttttaccca cataaagaaa atgatttaga ttgcaattct 1860
ttttattttt ctaaaagaat aaatatattc tctctttttt tttttaaaac aaaattctct 1920
ttcttacagg tatccagaaa atttttccct tatctggcag ctaattttaa cgatcctcgt 1980
gtaaccctag ttctcggaga tggtgcgtat atgatagtct cgttttatat tttatttcac 2040
ttgattttta ccttttttg tggttaatta atcatctacc attggttctc tttaccttca 2100
ggagctgcat ttgtaaaggc tgcacaagcg ggatattatg atgctattat agtggactct 2160
tctgatccca ttggtacgct attactattt aataccaaga ctattcttat tatataagct 2220
actaagaaac taattgaata attaataaac gtaactgtaa ttgatttcta aaataatata 2280
tataatttca ggtccagcaa aagatttgtt tgagaggcca ttctttgagg cagtagccaa 2340
agccccttagg ccaggaggg ttgtatgcac acaggctgaa agcatttggc ttcatatgca 2400
tattattaag caaatcattg ctaactgtcg tcaagtcttt aagggttctg tcaactatgc 2460
ttggacacc gttccaacat atcccacgta ttctttttct ctctctcttc ctgtcttttt 2520
cgatgcaatg taaatttata aaattggaag tccgttttac ttttctatag acgtagatcc 2580
taaaattgtc aagaaatgga gaattgactt acaagaaaaa tcaacttctt ttcatttact 2640
attctttttg gtgacaaact ttacttatta tttcgttcta aaatgaaaat ttattttat 2700
attttaaaat aatttagctt taaactttta attttacttg ttatattttt aataaaaaag 2760
atttattgtc aaataaatgt tgtggccata caataagttt caaattatgt cacaaaaatt 2820
gaaacagagt gagcaaattt ttatttcaag taaactttca aggaattgtg tttaagtttt 2880
ctcaactgat actgatcaat gatgtcttaa atattttgat ttcagcggtg tgatcggtta 2940
tatgctctgc tctactgaag ggccagaagt tgacttcaag aatccagtaa atccaattga 3000
caaagagaca actcaagtca agtccaaatt aggacctctc aagttctaca actctgatgt 3060
aacttcatat ctcacaattt ctttttccgt tttactgtat gttcttcatc aaattttata 3120
actaactctt ttcatattgt cttttttttt cagattcaca aagcagcatt cattttacca 3180
tctttcgcca gaagtatgat cgagtcttaa tcaagtgaat aatgaacact ggtcgtacaa 3240
tcattggacc aagatcaagt cttaatcaag tgaataaata agtgaaatgc aacgtattgt 3300
atgagaattc tgcagtaatt atcataattt ccaattcact aattgttgta aaattctttc 3360
tctgtggtgt ttggtacttt aatataaatt ttcctgctga agttttaaaa tggacgttcc 3420
aattcgatcc tcgcaaatca gttcattatc cttctttcag tgtactaaag ccaacaaatc 3480
tcatagttac cgtggtgttg tttttactat gggatttgcg atcttattaa ggttttggtc 3540
tatgttttgg aattgtaata ttcatatttt taagttactc tgtatctaaa tcggcctagt 3600
ctctcttttt taataaatgg cgtcatcgtc gttgcagttt gtttgctcat ttttcagaag 3660
tatgatggtt tgaaatgtct ttgtttctat ggcctcttta ctgcagctta ccgatttgct 3720
tatgttggat attgcttctg attcatatgg gatatgaagt ttggtatttg ttgaatgatt 3780
aaggaaaaaa tttacaacaa taactatgcc tcaatcccaa cctaattatg atcagcgctt 3840
caaaaaaaaa aaaaaagtag aactaaaatt tttaagctta gatgcaaatc cataacactg 3900
taaaaaaaag tagggatttc tgttcctttc ttttatagtt aagaaagaac tctctgcctt 3960
gtgaatctta ttgcatctct ctaattcagc aaaataattg aacaatgact aggataaaat 4020
atttcagcta agataaccct aattttctgg atggaatcac cttctccacc aagttgttat 4080
tttcttgcat tgtgagcaga ctagttcaat tacaccatta gagttttttc tcagcactat 4140
cctggcaatg atgcgctaag ctctgaaaac caattagcct taatagagtt ggagctcccc 4200
aataactctt 4210

<210> 3
<211> 3686
<212> DNA
<213> Nicotiana tabacum
<400> 3
ttgtgagaga tttggtgtcc tctacaatga ttgttgaagt ccctatttat agctataacac 60
aggaaacaaa atcctaggat caagcccctc ttaaatgaca ataatggggt taatgatgaa 120
tatgtagcgg catgacatga atgccaaaat tctccgcaac gactatttat ttaatattga 180
ggaatatttt ttattaaata ctatctggtg acaagcattc gtttgcttcc gttgattacg 240
ttgattttgg gatctactct ataccaaccg aagccgttgt ccttgatctt cgctttcatt 300
taattcatct tccgtctgcc tccgatttca caagtcatgc acccattcaa ttatttaatg 360
gaaaccaatt ttaccctata caaatggtac atcattcgtc aaatacttta cttggatata 420
aacaattttg cccgaggagt aaacagatgc gaagaaagaa agcagacgat taaagaaatt 480
tttaaaaaag gagagagaaa tgaacacaca catgtactaa taaaattagg gtactacttt 540
actaataatt ggacagagac taaattcata ttttagttcc aaaatgtctc gggcagtcca 600
accatgcacg ttgtaatgat tttttaactc tattatatcg agttgcgccc tccactcctc 660
ggtgtccaaa ttgtatataa atgcatatgt gtctattggg agtgtacatc aagctttcat 720
aaagtacaaa tcgtaatact tgttgaaaca taatactttc tcttctccaa tttgtttagt 780
ttaattttga aaatggaagt catatctacc aacacaaatg gctctaccat cttcaagagt 840
ggtgccattc ccatgaatgg ccaccataat ggcacttcca aacaccaaaa cggccacaag 900
aatgggactt ccgaacaaca gaacgggaca atcagccttg ataatggcaa cgagctactg 960
ggaaactcca attgtattaa gcctggttgg ttttcagagt ttagcgcatt atggccaggt 1020
tagtactgag aaagaaactc aaatgcatat ttaaagttaa aattgttagg ctaatataag 1080
gagttgatat tcttttagtg attaattaaa aaggaaaaag tatcaaataa attcaaaaaa 1140
tggatagtaa cttcgcatat tactctacac attaatttga aataaatcga attttgcagg 1200
tgaagcattc tcacttaagg ttgagaagtt actgttccag gggaagtctg actaccaaga 1260
tgtcatgctc tttgaggtaa ataatatttt aatacacatg cttccattta aattgatact 1320
tttaatttac ttttacttta ttgcatgtgt acgtacagtc agcaacttat gggaaggttc 1380
tgactttgga tggagcaatt caacacacag agaatggtgg atttccatac actgaaatga 1440
ttgttcatct tccacttggt tccatcccaa acccaaaaaa ggttttgatc atcggcggag 1500
gaattggttt tacattattc gaaatgcttc gttatcctac aatcgaaaaa attgacattg 1560
ttgagatcga tgacgtggta gttgatgtaa gtcaaacttc ttttactcac ataaaaaaat 1620
ggtttagatt gcttcttgtt atttttctaa aagaatacta tttttttaaa acaaaatttt 1680
cttttttaca ggtatctaga aaatttttcc cttatctcgc tgctaatttt aacgatcctc 1740
gtgtaaccct agtccttgga gatggtgcgt atttgataat ctcgcttttg ttttatcttt 1800
tatttttatt gcatttaatt tttacctttt ggtgtgtggt taattcacct gccattggtt 1860
ctctttcatt tcaggggctg catttgtaaa ggctgcacaa gcagaatatt atgatgctat 1920
tatagtggac tcttctgatc ccattggtac tctattactt cttaatacca agactaatct 1980
tattgaataa gctactaata aacggtaatt gatttctaaa acaatataat ttcaggtcca 2040
gcaaaagatt tgtttgagag gccattcttt gaggcagtag ctaaagccct aaggccagga 2100
ggagttgtat gcacacaggc tgaaagcatt tggcttcata tgcatattat taagcaaatc 2160
attgctaact gtcgtcaagt ctttaagggc tctgtcaact atgcttggac tactgttcca 2220
acatatccaa cgtatttttc tctctctctc tcttcctata aaattggaag ttttgattct 2280
ataattgtca agaaatggag aatcagttcc aagaaaaacc aacttctttt cttttactct 2340
tcaaggtatt gtgtttaatt ttttttcaac tgatatgatc aattattttg atttcagcgg 2400
tgtgattggt tatatgctct gctctactga aggaccagaa attgacttca agaatccagt 2460
aaatccaatt gacaaagaga cagctcaagt caagtccaaa ttagcacctc tcaagttcta 2520
caactctgat gtaacttcat atctcacaat ttcttttttc ctattgtact ttatgttctt 2580
cgtcaaattt tataattaac tcttttcaaa ttgtcttttt ttttttcaga ttcacaaagc 2640
agcattcatt ttgccatctt tcgccagaag tatgatcgag tcttaatcaa ctgattaatg 2700
aatactggtg gtacaatcat tggaccaaga tcaataagtg aaagacgtat tgtatgagaa 2760
ttctgcagta attaattatc ataatttcca atttaccaat tattgtaaaa ttctttctct 2820
gtggtgtttg gtactttaat ataaattttc ctgcttaagt tttgaatcga cgtatcaact 2880
caatcctcgc aaatcacttc attaccctcc tttcagtgta ctaaagtaaa ctgttgcgga 2940
atatcgtggg ttaactagaa cacaatcaca cacaaatcaa atagagaaga atttattaac 3000
ggggttcggc taagcctaat cctcaggaca aaagcagaga gagttttcca ctatgaatga 3060
gaagaaaaac acaatacaat atataaaatc ctcaactaca acccctatat atgatcccaa 3120
aaggtcccaa acatatatga gaaaagtttc ccaatttgac aaagattata ggttttcctt 3180
tcccaaatct attagggcaa tgggttttct taaacctatg gggactatgg ctttcctaaa 3240
aatacaagga aataattcaa accacaaata ataaatcttc cccttggctt gaattctctt 3300
catcaacagg aacaatagct ctctaccttg ccctcaaccc tcgcaagggc tctattgatt 3360
gtcgcacaca tcaaccaagt ctaggcaacg cctaaacttg ttatgagact taatctcttc 3420
agccatagca ctaatccgtc gatttggttc tgtacccgaa atagcttttg gatagctata 3480
acactgaaac acatcagtgg tctctgcaac taccaaagca aatcccacag tattcgtata 3540
tccaagaaga tttccatcaa ctacgtttgc aaacctttgc ggtcggttga tcactctctt 3600
ctctctgtat gttgcaatgc tatatggttg ttgttgcgca ggtgcatcaa cattatcgtc 3660
ttgatcaata ttttgtacct ccctta 3686

<210> 4
<211> 4035
<212> DNA
<213> Nicotiana tabacum
<400> 4
ggaagagtgt ggtatgggag atgcctccca gggagtacct aaagctgaat actgatggaa 60
gttttaacaa acaaattggg aaagcaggga ttggagggat tctcagagat gaagagggag 120
gctttgtcat ggctttttcg atgcctataa tctataataa catcagtgaa gcagaattga 180
aagccatcaa gtatgggtgt gaatggtgca aatacaaagg aatatcaaac ttcattgtgg 240
aaactgactc gaggatgatc tatgacatac tacagaccaa aaatctaagc aacaacaagt 300
tgaaacaaga gaccgagaaa ttaatggaga ttctggacac ctgcaggaca cctgttaccc 360
attgccttcg cgaagcaaat caagtggcag actggtttgc taaagaggcc accagagcta 420
acgaaggtat cactcataca gattttagac aggtatcaaa agcggccaag ggccctttct 480
tcatggatat gtggcaggtc ccttatttta gaattagata tgaaaaatct aatttttttt 540
tgtaagttaa ttctgtgtat agtgagagga aatcgtctaa tatgtatttt tgcccataga 600
ctcttcctct ccttaggtaa aaaggtagct ccgaggtaag gtttatgttc ccctcagtgt 660
aacctttttt tgtttatata atagacatgg tatgggtcca gctaaaccccc caacaccaca 720
ggggatagat acctgggtga ttggtttatt ttttaaaaaa aaaaacttta ctaataattg 780
cacggagaca aaacttatat tttagttcca aaatgacagt ccaaccatgc acgttgtaat 840
gattttttaa ctctattata tcgagttccg ccctccactc ctcggtgtcc aaattgtatt 900
taaatgcata gatatgttta ttgggagtgt acatcaagct ttcagaaaat acaaaccata 960
atactttctc ttctccaatt tgcttagttt aatttggaaa atggaagtca tatctaccaa 1020
cacaaatggc tctactatct tcaagaatgg tgccattccc atgaacggtt accagaatgg 1080
cacttccaaa caccaaaacg gccaccagaa tggcacttcc gaacatcgga acggccacca 1140
gaatgggatt tccgaacacc aaaacggcca ccagaatggc acttccgagc atcagaacgg 1200
ccatcagaat gggacaatca gccatgacaa cggcaacgag ctacagctac tgggaagctc 1260
caactctatt aagcctggtt ggttttcaga gtttagcgca ttatggccag gttagtacta 1320
agaaagaaac tcaaatgcat cgtactcttg tattctgctt tgcgtataat ttagatgatg 1380
gtgtttgact aagcactgag tttaaaaata aaaagtttaa agttaaattg ttactataga 1440
gagctatatc tttaggaact aactaaaaag gaaaaattat cacataaaat tgggatgaag 1500
taagcagtta acttcgcata ttattcgaca cattaatttg aaataaatcg aattttgcag 1560
gtgaagcatt ctcacttaag gttgagaagt tactattcca ggggaagtct gattaccaag 1620
atgtcatgct ctttgaggta attaattaat actaatagtc aagctcatgt atgattatat 1680
ttaaagtggt atttttcgtt tatttttaat ttattgcacg tgtacgtaca gtcagcaaca 1740
tatgggaagg ttctgacttt ggatggagca attcaacaca cagagaatgg tggatttcca 1800
tacactgaaa tgattgttca tcttccactt ggttccatcc caaaccctaa aaaggttttg 1860
atcatcggcg gaggaattgg ttttacatta ttcgaaatgc ttcgttatcc tacaatcgaa 1920
aaaattgaca ttgttgagat cgatgacgtg gtagttgatg taagtcaaac ttcttttact 1980
cacataaaaa aatgatttag attcttattt ttctaaaaga attaaaacaa aattttccgt 2040
tttacaggta tctagaaaat ttttccctta tcttgctgct aattttagcg atcctcgtgt 2100
aaccctagtc cttggagatg gtgcgtattt gataatctcg tttttatttt atcttttact 2160
tttattttat ttaattttta cctttttgtg tgtggttaat tcacctgcca ttggttcttt 2220
ttatttcagg ggctgcattt gtaaaggccg cacaagcagg atattatgat gctattatag 2280
tggactcttc tgatcccatt ggtactctat tactacttaa taccaagact attcttatta 2340
aataagctac taataaacgt aactctgata gttttctaaa ataatataat ttcaggtcca 2400
gcaaaagact tgtttgagag gccattcttt gaggcagtag ccaaagccct aaggccagga 2460
ggagttgtat gcacacaggc tgaaagcatt tggcttcata tgcatattat taagcaaatc 2520
attgctaact gtcgtcaagt ctttaagggc tctgtcaact atgcttggac tactgttcca 2580
acatatccaa cgtatttttc tctctctctt cctataaaat tggaagtttt gattctataa 2640
ttgtcaagaa atggagaatc agttccaaga aaaaccaaat tcttttcttt tactcttcaa 2700
ggtgtgttta agttttttaa actgatactg atcaattatt ttgatttcag cggtgtgatt 2760
ggttatatgc tctgttctac tgaaggacca gaagttgact tcaagaatcc agtaaatcca 2820
attgacaaag agacaactca agtcaagtcc aaattagcac ctctcaagtt ctacaactct 2880
gatgtaactt catatctcaa tttctttttt cttatgtac tttatgttct tagtcaaatt 2940
ttataattaa ctcttttcaa attgtctttt tttttcagat tcacaaagca gcattcattt 3000
tgccatcttt cgccagaagt atgatcgagt cttaatcaag tgactaatga atactggcgg 3060
tacaatcatt ggaccaagat cgagtcttaa tcaagtgaat aaataagtga aatgcgacgt 3120
attgtataag aattctgcag tagttaatta tcataatttc caattcacca attactgtaa 3180
aattctttct ctgtggtgtt tggtacttta ctataaattt tcccgcttaa gttttgaatc 3240
gacgtttcaa ctcagtcctc gcaaatcact tcattaccct tctttcggta tactaaagta 3300
aactgttgca gaatatcgtg ggttaactag aacacaatca cacacatagc aaatagagaa 3360
gaaaaatcaa cacaagaatt tattaacgag gttcggctaa gcctaatcct ccgggcaaaa 3420
gcagagagag tttttcacta tgaatgagaa gtaaaacaca atacaatcta tagaatttcc 3480
aactacaacc cctatata gatttcaaaa ggtcccaaac atatatgaga aaggtttccc 3540
aatttgacaa ggattatagg ttttcttttc caaaatctat tagggtaatg ggttttccta 3600
aacctatgag gactatgggt ttcctaaaaa tacaaggaaa taatttaaac caaaaataac 3660
aaatcttccc cttggtttga attctcttca ttaacaggaa caacagctct ctacctttcc 3720
ctcagccctc gcaatggctc tattgattgc cgcactcatc aaccaagtct acgcaacgcc 3780
taaacttgtt aagagactta atctcttcag ccatagcact aatttgtcga tttggttctg 3840
tacccgaaat agcttttgga taactataac actcaaatgc atcagtggtc tctgcaacta 3900
ccaaagcaaa tcccacaaga ttcgtatatc caagaagatt tccatcaact acatttgcaa 3960
acctttgcgg tcggttgatc actctctctc tctctgcatg ttgcaatgct atatggttgt 4020
tgttgtgcat gtgca 4035

<210> 5
<211> 4431
<212> DNA
<213> Nicotiana tabacum
<400> 5
acggtacaat tgaatttgtt gcgtgacttg tagacaagtg aattgatttg tccaaaatga 60
ttaataaatc aaattaaaaa taaggcttag cgttaaaatc aaaggaaatg gcaagcctga 120
ctcccggagc aatgcttctg aggacagtag taaaaacaat atcagacaaa aagtaaagtt 180
atattattta gcttgaggat aaagtatgtc atcagttttg ttagagattt ggtgtcctct 240
acaatgattg ttgaagtccc tatttatagc tatacatagg aaacaagatc ctagaatcaa 300
acccttctta aatgacatta atgggagtta ttgatgaata tgtagcggca tgacatgaat 360
gccaaaattc tccgcaacgg ctatttactt aatattgagg aatatttttc attaaatact 420
atctggtgac aagtattcgt ttgcttccgt tgattgcatt gattttggga tctactatgt 480
accaaccgaa gttgttgtcc ttgatcttcg ctttcattta attcatcttt cgtcgacctc 540
tgattccaca agtcatgcac ccattcaatt atttaatgga aaccaatttt accctgtaca 600
aatggtacaa atactttcct tggataaaaa caattttgcc taaggagtaa acagatgcga 660
agtaagaaag cagacgacta aagaaaattt taaaaaagga gagagaaatg agcacacaca 720
cgtactaata aaattagggt actactttac taataattgg acagagacta aattcatatt 780
ttagttccaa aatgtctcgg gcagtccaac catgcacgtt gtaatgagtt tttaactcta 840
ttatctcgag ttgcgccctc cactcctctg tgtccaagtt gtatataaat gcatatatgt 900
ctattgggag tgtacagcga gctttcataa agtacaaatc ataatacttg ttgaaacata 960
atactttctc ttctccaatt tgtttagttt aattttgaaa atggaagtca tatctaccaa 1020
cacaaatggc tcgaccatct tcaagaatgg tgccattccc atgaatggcc accagagtgg 1080
cacttccaaa cacctcaacg gctaccagaa cggcacttcc aaacaccaaa acggccacca 1140
taatggcact tccgaacatc ggaacggcca ccagaatggg atttccgaac accaaaacgg 1200
ccaccagaat gggacttccg aacatcggaa cggccaccag aatgggattt ccgaacacca 1260
aaacggccac cagaatggga cttccgaaca ccaaaacggc caccagaatg ggacttccga 1320
acaacagaac gggacaatca gccatgacaa tggcaacgag ctactgggaa actccaactc 1380
tattaagctt ggttggtttt cagagtttag cgcattatgg ccaggttagt actgagaaag 1440
aaactcaaat tcatatttaa agttaaaatt gttaggctaa tataagaagt tgattttctt 1500
ttagtgatta attaaaaaag gaaagagtat caaataaatt ccaaaaaatg accagtaact 1560
tcgcatatta ttctacacat taatttgaaa taaatcgaat tttgcaggtg aagcattctc 1620
ccttaaggtt gagaagttac tatttcaggg gaagtctgac taccaagatg tcatgctctt 1680
tgaggtaaat aatattctaa tacacatgct ttaatatgaa taaatacttt taatttactt 1740
ttagtttatt gcacgtgtac gtacagtcag caacatatgg gaaggttttg actttggatg 1800
gagcaattca acacagag aatggtggat ttccatacac tgaaatgatt gttcatcttc 1860
cacttggttc catcccaaac ccaaaaaagg ttttgatcat cggcggagga attggtttta 1920
cattattcga aatgcttcgt tatcctacaa tcgaaaaaat tgacattgtt gaaatcgatg 1980
acgtggtagt tgatgtaagt caaatttctt ttactcacat aaaaaaatga tttagattgc 2040
ttctttttat ttttctaaaa gaataaatat attctctctt agttttaaac aaaattctct 2100
ttcttacagg tatctagaaa atctttccct tatctcgcag ctaattttaa tgatcctcgt 2160
gtaaccctcg ttctcggaga tggtgcgtat ttataatctc gtttttgttt tatcttttat 2220
ttttatttca tttaatttac ctttttgtgt gtggttaatt tacccgtcat tggttctctt 2280
tcatttcagg ggctgcattt gtaaaggctg cacaagcagg atattatgat gctattatag 2340
tggactcttc tgatcccatt ggtactctat tactacttaa taccaagact aatcttattg 2400
aataagctac taataaactg taattgattt ctaaaataat ataatttcag gtccagcaaa 2460
agatttgttt gagaggccat tctttgaggc agtagccaaa gccctaaggc caggaggagt 2520
tgtatgcaca caggccgaaa gcatttggct tcatatgcat attattaagc aaatcattgc 2580
taactgtcgt caagtcttta agggctctgt caactacgct tggactactg ttccaacata 2640
tcccagtat tttctctctc tctctcttca tctttgaaaa ttgaaaatcc tgactacttt 2700
ccttcctttg attcctcggt taaaggggcg tagatcataa gattttcaag aaatagataa 2760
tgacgtccaa gaaaaactaa cttcttttca tttactattc tttttggtga caaactttat 2820
ttattatttc gttctaaaga gaaaatttat ttttatattt taaaataatt ttgttttaaa 2880
cttttattt tacttattat atctttaata aaaaaattat agtcaaataa atattatggc 2940
cacactaaac atccaagttt ttgaaaccat aagttttaga gccaaatgag ttaatttgtt 3000
tttggtatgc gggtgcggag tcaaattatg tcacaaaaat tgtaatggag tgagcaaatt 3060
tttatttcga gtaaactttc aaggtattgt gttaaagttt tttcaactga tactaatcaa 3120
ttatgtctca accattttga tttcagtggt gtaattgggt atatgctctg ctctactgaa 3180
gggccagaag ttgacttcaa gaatccaata aatccaattg acaaagagac aactcaagtc 3240
aagtccaaat tagcacctct caagttttac aattctgatg taacttcata tctaacaatt 3300
tctttttctg ttttactgta tcttcattgt caaaatttta taattaactc ttctcaaatt 3360
atcttttttt ttagattcac aaagcagcat tcattttgcc atctttcgcc agaagtatga 3420
tcgagtctta atcaagtgaa taatgaacac tggtggtgca atcattggac caagatcgag 3480
tcttaatcaa gtgaataaat aagtgaaatg ccgacgtatt gtatgagaat tctacagtaa 3540
ttaattatca taatttccaa ttcaccaatt attgtaaaat tctttctctg tggtgtttgg 3600
tacttcaata taaattttcc tgctgaagtt ttgaatcgat gttccaactc aatcctcgca 3660
aatcagttcc ttatttttct ttcagtgtac ttagtaatct tagtgggatc ggcgcttcca 3720
taaaagaatg taaacttaat atttttttta agcctaaatg caaatccatc acactgtaaa 3780
acatattgat cttcagcctc tgaagatgag tagggatttc tgctcctttc atttacagtc 3840
gagaactttc tgtcttgcat ctctctaatt cagccaaatt attgataacc acgactttag 3900
ttaaacattt gtttaactaa aacaactccc ctaaccatgg taaaacacag tagttatcag 3960
tgtttaatta acctttcctt tttggtaaat atagggtgaa gtcttgctgc gaactttgtt 4020
ctcatatgtt gaagatagta atccaactca agaaacaaac atgattgatt aaatgttgtc 4080
gatgacacac tttcaactga tccaaatggt aacgttccgg ccggctattt tatgtatttg 4140
agtcccgttc tcctacttga tattttccct atgcttgttt gacgttttgt gagttgtgga 4200
gatggttggt ttggttcggg aaggttttgg aatgagtaga gacgcttcaa ctcattttga 4260
aagcttaagt tgtacgagtt gatcaaggtt tgactttggt ataaacgata tcaatttgat 4320
ggtttcagta tgttcatatg gtgattttgg aattagacgc atgttcggat atgaatttgg 4380
aggtttctag aatgttttgt ctctgagtgc caaaagctgg gaatttaaag g 4431

<210> 6
<211> 1128
<212> DNA
<213> Nicotiana tabacum
<400> 6
atggaagtca tatctaccaa cacaaatggc tctaccatct tcaagaatgg taccattccc 60
atgaacggcc accaaaatgg ctcttccgaa cacctcaacg gctaccagaa tggcatttcc 120
aaacaccaaa acgggcacca gaatggcact tccgaacatc ggaacggcca ccagaatggg 180
acatccgaac aacagaacgg gacaatcagc catgacaatg gcaacgagct actgggaagc 240
tccaactcta ttaagcctgg ttggttttca gagtttagcg cattatggcc aggtgaagca 300
ttctcactta aggtcgagaa gttactattc caggggaaat ctgattacca agatgtcatg 360
ctctttgagt cagcaactta tgggaaggtt ctgactttgg atggagcaat tcaacataca 420
gagaatggtg gatttccata cactgaaatg attgttcatc taccacttgg ttccatccca 480
aaccccaaaaa aggttttgat catcggcgga ggaattggtt ttacattatt cgaaatgctt 540
cgttatcctt caatcgaaaa aattgacatt gttgagatcg atgacgtggt agttgatgta 600
tccagaaaat ttttccctta tctggcagct aattttaacg atcctcgtgt aaccctagtt 660
ctcggagatg gagctgcatt tgtaaaggct gcacaagcgg gatattatga tgctattata 720
gtggactctt ctgatcccat tggtccagca aaagatttgt ttgagaggcc attctttgag 780
gcagtagcca aagcccttag gccagggagga gttgtatgca cacaggctga aagcatttgg 840
cttcatatgc atattattaa gcaaatcatt gctaactgtc gtcaagtctt taagggttct 900
gtcaactatg cttggacaac cgttccaaca tatcccaccg gtgtgattgg ttatatgctc 960
tgctctactg aagggccaga agttaacttc aagaatccag taaatccaat tgacaaagag 1020
acaactcaag tcaagtccaa attaggacct ctcaagttct acaactctga tattcacaaa 1080
gcagcattca ttttgccatc tttcgcccga agtatgatcg agtcttaa 1128

<210> 7
<211> 1128
<212> DNA
<213> Nicotiana tabacum
<400> 7
atggaagtca tatctaccaa cacaaatggc tctaccatct tcaagaatgg tgccattccc 60
atgaacggcc accaaaatgg cacttctgaa cacctcaacg gctaccagaa tggcacttcc 120
aaacaccaaa acgggcacca gaatggcact ttcgaacatc ggaacggcca ccagaatggg 180
acatccgaac aacagaacgg gacaatcagc catgacaatg gcaacgagct actgggaagc 240
tccgactcta ttaagcctgg ctggttttca gagtttagcg cattatggcc aggtgaagca 300
ttctcactta aggttgagaa gttactattc caggggaagt ctgattacca agatgtcatg 360
ctctttgagt cagcaactta tgggaaggtt ctgactttgg atggagcaat tcaacataca 420
gagaatggtg gatttccata cactgaaatg attgttcatc taccacttgg ttccatccca 480
aaccccaaaaa aggttttgat catcggcgga ggaattggtt ttacattatt cgaaatgctt 540
cgttatcctt caatcgaaaa aattgacatt gttgagatcg atgacgtggt agttgatgta 600
tccagaaaat ttttccctta tctggcagct aattttaacg atcctcgtgt aaccctagtt 660
ctcggagatg gagctgcatt tgtaaaggct gcacaagcgg gatattatga tgctattata 720
gtggactctt ctgatcccat tggtccagca aaagatttgt ttgagaggcc attctttgag 780
gcagtagcca aagcccttag gccagggagga gttgtatgca cacaggctga aagcatttgg 840
cttcatatgc atattattaa gcaaatcatt gctaactgtc gtcaagtctt taagggttct 900
gtcaactatg cttggacaac cgttccaaca tatcccaccg gtgtgatcgg ttatatgctc 960
tgctctactg aagggccaga agttgacttc aagaatccag taaatccaat tgacaaagag 1020
acaactcaag tcaagtccaa attaggacct ctcaagttct acaactctga tattcacaaa 1080
gcagcattca ttttaccatc tttcgccaga agtatgatcg agtcttaa 1128

<210> 8
<211> 1062
<212> DNA
<213> Nicotiana tabacum
<400> 8
atggaagtca tatctaccaa cacaaatggc tctaccatct tcaagagtgg tgccattccc 60
atgaatggcc accataatgg cacttccaaa caccaaaacg gccacaagaa tgggacttcc 120
gaacaacaga acgggacaat cagccttgat aatggcaacg agctactggg aaactccaat 180
tgtattaagc ctggttggtt ttcagagttt agcgcattat ggccaggtga agcattctca 240
cttaaggttg agaagttact gttccagggg aagtctgact accaagatgt catgctcttt 300
gagtcagcaa cttatgggaa ggttctgact ttggatggag caattcaaca cacagagaat 360
ggtggatttc catacactga aatgattgtt catcttccac ttggttccat cccaaaccca 420
aaaaaggttt tgatcatcgg cggaggaatt ggttttacat tattcgaaat gcttcgttat 480
cctacaatcg aaaaaattga cattgttgag atcgatgacg tggtagttga tgtatctaga 540
aaatttttcc cttatctcgc tgctaatttt aacgatcctc gtgtaaccct agtccttgga 600
gatggggctg catttgtaaa ggctgcacaa gcagaatatt atgatgctat tatagtggac 660
tcttctgatc ccattggtcc agcaaaagat ttgtttgaga ggccattctt tgaggcagta 720
gctaaagccc taaggccagg aggagttgta tgcacacagg ctgaaagcat ttggcttcat 780
atgcatatta ttaagcaaat cattgctaac tgtcgtcaag tctttaaggg ctctgtcaac 840
tatgcttgga ctactgttcc aacatatcca accggtgtga ttggttatat gctctgctct 900
actgaaggac cagaaattga cttcaagaat ccagtaaatc caattgacaa agagacagct 960
caagtcaagt ccaaattagc acctctcaag ttctacaact ctgatattca caaagcagca 1020
ttcattttgc catctttcgc cagaagtatg atcgagtctt aa 1062

<210> 9
<211> 1146
<212> DNA
<213> Nicotiana tabacum
<400> 9
atggaagtca tatctaccaa cacaaatggc tctactatct tcaagaatgg tgccattccc 60
atgaacggtt accagaatgg cacttccaaa caccaaaacg gccaccagaa tggcacttcc 120
gaacatcgga acggccacca gaatgggatt tccgaacacc aaaacggcca ccagaatggc 180
acttccgagc atcagaacgg ccatcagaat gggacaatca gccatgacaa cggcaacgag 240
ctacagctac tgggaagctc caactctatt aagcctggtt ggttttcaga gtttagcgca 300
ttatggccag gtgaagcatt ctcacttaag gttgagaagt tactattcca ggggaagtct 360
gattaccaag atgtcatgct ctttgagtca gcaacatatg ggaaggttct gactttggat 420
ggagcaattc aacacacaga gaatggtgga tttccataca ctgaaatgat tgttcatctt 480
ccacttggtt ccatcccaaa ccctaaaaag gttttgatca tcggcggagg aattggtttt 540
acattattcg aaatgcttcg ttatcctaca atcgaaaaaa ttgacattgt tgagatcgat 600
gacgtggtag ttgatgtatc tagaaaattt ttccccttatc ttgctgctaa ttttagcgat 660
cctcgtgtaa ccctagtcct tggagatggg gctgcatttg taaaggccgc acaagcagga 720
tattatgatg ctattatagt ggactcttct gatcccattg gtccagcaaa agacttgttt 780
gagaggccat tctttgaggc agtagccaaa gccctaaggc caggaggagt tgtatgcaca 840
caggctgaaa gcatttggct tcatatgcat attattaagc aaatcattgc taactgtcgt 900
caagtcttta agggctctgt caactatgct tggactactg ttccaacata tccaaccggt 960
gtgattggtt atatgctctg ttctactgaa ggaccagaag ttgacttcaa gaatccagta 1020
aatccaattg acaaagagac aactcaagtc aagtccaaat tagcacctct caagttctac 1080
aactctgata ttcacaaagc agcattcatt ttgccatctt tcgccagaag tatgatcgag 1140
tcttaa 1146

<210> 10
<211> 1260
<212> DNA
<213> Nicotiana tabacum
<400> 10
atggaagtca tatctaccaa cacaaatggc tcgaccatct tcaagaatgg tgccattccc 60
atgaatggcc accagagtgg cacttccaaa cacctcaacg gctaccagaa cggcacttcc 120
aaacaccaaa acggccacca taatggcact tccgaacatc ggaacggcca ccagaatggg 180
atttccgaac accaaaacgg ccaccagaat gggacttccg aacatcggaa cggccaccag 240
aatgggattt ccgaacacca aaacggccac cagaatggga cttccgaaca ccaaaacggc 300
caccagaatg ggacttccga acaacagaac gggacaatca gccatgacaa tggcaacgag 360
ctactgggaa actccaactc tattaagctt ggttggtttt cagagtttag cgcattatgg 420
ccaggtgaag cattctccct taaggttgag aagttactat ttcaggggaa gtctgactac 480
caagatgtca tgctctttga gtcagcaaca tatgggaagg ttttgacttt ggatggagca 540
attcaacaca cagagaatgg tggatttcca tacactgaaa tgattgttca tcttccactt 600
ggttccatcc caaaccccaaa aaaggttttg atcatcggcg gaggaattgg ttttacatta 660
ttcgaaatgc ttcgttatcc tacaatcgaa aaaattgaca ttgttgaaat cgatgacgtg 720
gtagttgatg tatctagaaa atctttccct tatctcgcag ctaattttaa tgatcctcgt 780
gtaaccctcg ttctcggaga tggggctgca tttgtaaagg ctgcacaagc aggatattat 840
gatgctatta tagtggactc ttctgatccc attggtccag caaaagattt gtttgagagg 900
ccattctttg aggcagtagc caaagcccta aggccaggag gagttgtatg cacacaggcc 960
gaaagcattt ggcttcatat gcatattatt aagcaaatca ttgctaactg tcgtcaagtc 1020
tttaagggct ctgtcaacta cgcttggact actgttccaa catatcccac tggtgtaatt 1080
gggtatatgc tctgctctac tgaagggcca gaagttgact tcaagaatcc aataaatcca 1140
attgacaaag agacaactca agtcaagtcc aaattagcac ctctcaagtt ttacaattct 1200
gatattcaca aagcagcatt cattttgcca tctttcgcca gaagtatgat cgagtcttaa 1260

<210> 11
<211> 375
<212> PRT
<213> Nicotiana tabacum
<400> 11
Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn
1 5 10 15
Gly Thr Ile Pro Met Asn Gly His Gln Asn Gly Ser Ser Glu His Leu
20 25 30
Asn Gly Tyr Gln Asn Gly Ile Ser Lys His Gln Asn Gly His Gln Asn
35 40 45
Gly Thr Ser Glu His Arg Asn Gly His Gln Asn Gly Thr Ser Glu Gln
50 55 60
Gln Asn Gly Thr Ile Ser His Asp Asn Gly Asn Glu Leu Leu Gly Ser
65 70 75 80
Ser Asn Ser Ile Lys Pro Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp
85 90 95
Pro Gly Glu Ala Phe Ser Leu Lys Val Glu Lys Leu Leu Phe Gln Gly
100 105 110
Lys Ser Asp Tyr Gln Asp Val Met Leu Phe Glu Ser Ala Thr Tyr Gly
115 120 125
Lys Val Leu Thr Leu Asp Gly Ala Ile Gln His Thr Glu Asn Gly Gly
130 135 140
Phe Pro Tyr Thr Glu Met Ile Val His Leu Pro Leu Gly Ser Ile Pro
145 150 155 160
Asn Pro Lys Lys Val Leu Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu
165 170 175
Phe Glu Met Leu Arg Tyr Pro Ser Ile Glu Lys Ile Asp Ile Val Glu
180 185 190
Ile Asp Asp Val Val Val Asp Val Ser Arg Lys Phe Phe Pro Tyr Leu
195 200 205
Ala Ala Asn Phe Asn Asp Pro Arg Val Thr Leu Val Leu Gly Asp Gly
210 215 220
Ala Ala Phe Val Lys Ala Ala Gln Ala Gly Tyr Tyr Asp Ala Ile Ile
225 230 235 240
Val Asp Ser Ser Asp Pro Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg
245 250 255
Pro Phe Phe Glu Ala Val Ala Lys Ala Leu Arg Pro Gly Gly Val Val
260 265 270
Cys Thr Gln Ala Glu Ser Ile Trp Leu His Met His Ile Ile Lys Gln
275 280 285
Ile Ile Ala Asn Cys Arg Gln Val Phe Lys Gly Ser Val Asn Tyr Ala
290 295 300
Trp Thr Thr Val Pro Thr Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu
305 310 315 320
Cys Ser Thr Glu Gly Pro Glu Val Asn Phe Lys Asn Pro Val Asn Pro
325 330 335
Ile Asp Lys Glu Thr Thr Gln Val Lys Ser Lys Leu Gly Pro Leu Lys
340 345 350
Phe Tyr Asn Ser Asp Ile His Lys Ala Ala Phe Ile Leu Pro Ser Phe
355 360 365
Ala Arg Ser Met Ile Glu Ser
370 375

<210> 12
<211> 375
<212> PRT
<213> Nicotiana tabacum
<400> 12
Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn
1 5 10 15
Gly Ala Ile Pro Met Asn Gly His Gln Asn Gly Thr Ser Glu His Leu
20 25 30
Asn Gly Tyr Gln Asn Gly Thr Ser Lys His Gln Asn Gly His Gln Asn
35 40 45
Gly Thr Phe Glu His Arg Asn Gly His Gln Asn Gly Thr Ser Glu Gln
50 55 60
Gln Asn Gly Thr Ile Ser His Asp Asn Gly Asn Glu Leu Leu Gly Ser
65 70 75 80
Ser Asp Ser Ile Lys Pro Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp
85 90 95
Pro Gly Glu Ala Phe Ser Leu Lys Val Glu Lys Leu Leu Phe Gln Gly
100 105 110
Lys Ser Asp Tyr Gln Asp Val Met Leu Phe Glu Ser Ala Thr Tyr Gly
115 120 125
Lys Val Leu Thr Leu Asp Gly Ala Ile Gln His Thr Glu Asn Gly Gly
130 135 140
Phe Pro Tyr Thr Glu Met Ile Val His Leu Pro Leu Gly Ser Ile Pro
145 150 155 160
Asn Pro Lys Lys Val Leu Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu
165 170 175
Phe Glu Met Leu Arg Tyr Pro Ser Ile Glu Lys Ile Asp Ile Val Glu
180 185 190
Ile Asp Asp Val Val Val Asp Val Ser Arg Lys Phe Phe Pro Tyr Leu
195 200 205
Ala Ala Asn Phe Asn Asp Pro Arg Val Thr Leu Val Leu Gly Asp Gly
210 215 220
Ala Ala Phe Val Lys Ala Ala Gln Ala Gly Tyr Tyr Asp Ala Ile Ile
225 230 235 240
Val Asp Ser Ser Asp Pro Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg
245 250 255
Pro Phe Phe Glu Ala Val Ala Lys Ala Leu Arg Pro Gly Gly Val Val
260 265 270
Cys Thr Gln Ala Glu Ser Ile Trp Leu His Met His Ile Ile Lys Gln
275 280 285
Ile Ile Ala Asn Cys Arg Gln Val Phe Lys Gly Ser Val Asn Tyr Ala
290 295 300
Trp Thr Thr Val Pro Thr Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu
305 310 315 320
Cys Ser Thr Glu Gly Pro Glu Val Asp Phe Lys Asn Pro Val Asn Pro
325 330 335
Ile Asp Lys Glu Thr Thr Gln Val Lys Ser Lys Leu Gly Pro Leu Lys
340 345 350
Phe Tyr Asn Ser Asp Ile His Lys Ala Ala Phe Ile Leu Pro Ser Phe
355 360 365
Ala Arg Ser Met Ile Glu Ser
370 375

<210> 13
<211> 353
<212> PRT
<213> Nicotiana tabacum
<400> 13
Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Ser
1 5 10 15
Gly Ala Ile Pro Met Asn Gly His His Asn Gly Thr Ser Lys His Gln
20 25 30
Asn Gly His Lys Asn Gly Thr Ser Glu Gln Gln Asn Gly Thr Ile Ser
35 40 45
Leu Asp Asn Gly Asn Glu Leu Leu Gly Asn Ser Asn Cys Ile Lys Pro
50 55 60
Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp Pro Gly Glu Ala Phe Ser
65 70 75 80
Leu Lys Val Glu Lys Leu Leu Phe Gln Gly Lys Ser Asp Tyr Gln Asp
85 90 95
Val Met Leu Phe Glu Ser Ala Thr Tyr Gly Lys Val Leu Thr Leu Asp
100 105 110
Gly Ala Ile Gln His Thr Glu Asn Gly Gly Phe Pro Tyr Thr Glu Met
115 120 125
Ile Val His Leu Pro Leu Gly Ser Ile Pro Asn Pro Lys Lys Val Leu
130 135 140
Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu Phe Glu Met Leu Arg Tyr
145 150 155 160
Pro Thr Ile Glu Lys Ile Asp Ile Val Glu Ile Asp Asp Val Val Val
165 170 175
Asp Val Ser Arg Lys Phe Phe Pro Tyr Leu Ala Ala Asn Phe Asn Asp
180 185 190
Pro Arg Val Thr Leu Val Leu Gly Asp Gly Ala Ala Phe Val Lys Ala
195 200 205
Ala Gln Ala Glu Tyr Tyr Asp Ala Ile Ile Val Asp Ser Ser Asp Pro
210 215 220
Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg Pro Phe Phe Glu Ala Val
225 230 235 240
Ala Lys Ala Leu Arg Pro Gly Gly Val Val Cys Thr Gln Ala Glu Ser
245 250 255
Ile Trp Leu His Met His Ile Ile Lys Gln Ile Ile Ala Asn Cys Arg
260 265 270
Gln Val Phe Lys Gly Ser Val Asn Tyr Ala Trp Thr Thr Val Pro Thr
275 280 285
Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu Cys Ser Thr Glu Gly Pro
290 295 300
Glu Ile Asp Phe Lys Asn Pro Val Asn Pro Ile Asp Lys Glu Thr Ala
305 310 315 320
Gln Val Lys Ser Lys Leu Ala Pro Leu Lys Phe Tyr Asn Ser Asp Ile
325 330 335
His Lys Ala Ala Phe Ile Leu Pro Ser Phe Ala Arg Ser Met Ile Glu
340 345 350
Ser

<210> 14
<211> 381
<212> PRT
<213> Nicotiana tabacum
<400> 14
Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn
1 5 10 15
Gly Ala Ile Pro Met Asn Gly Tyr Gln Asn Gly Thr Ser Lys His Gln
20 25 30
Asn Gly His Gln Asn Gly Thr Ser Glu His Arg Asn Gly His Gln Asn
35 40 45
Gly Ile Ser Glu His Gln Asn Gly His Gln Asn Gly Thr Ser Glu His
50 55 60
Gln Asn Gly His Gln Asn Gly Thr Ile Ser His Asp Asn Gly Asn Glu
65 70 75 80
Leu Gln Leu Leu Gly Ser Ser Asn Ser Ile Lys Pro Gly Trp Phe Ser
85 90 95
Glu Phe Ser Ala Leu Trp Pro Gly Glu Ala Phe Ser Leu Lys Val Glu
100 105 110
Lys Leu Leu Phe Gln Gly Lys Ser Asp Tyr Gln Asp Val Met Leu Phe
115 120 125
Glu Ser Ala Thr Tyr Gly Lys Val Leu Thr Leu Asp Gly Ala Ile Gln
130 135 140
His Thr Glu Asn Gly Gly Phe Pro Tyr Thr Glu Met Ile Val His Leu
145 150 155 160
Pro Leu Gly Ser Ile Pro Asn Pro Lys Lys Val Leu Ile Ile Gly Gly
165 170 175
Gly Ile Gly Phe Thr Leu Phe Glu Met Leu Arg Tyr Pro Thr Ile Glu
180 185 190
Lys Ile Asp Ile Val Glu Ile Asp Asp Val Val Val Asp Val Ser Arg
195 200 205
Lys Phe Phe Pro Tyr Leu Ala Ala Asn Phe Ser Asp Pro Arg Val Thr
210 215 220
Leu Val Leu Gly Asp Gly Ala Ala Phe Val Lys Ala Ala Gln Ala Gly
225 230 235 240
Tyr Tyr Asp Ala Ile Ile Val Asp Ser Ser Asp Pro Ile Gly Pro Ala
245 250 255
Lys Asp Leu Phe Glu Arg Pro Phe Phe Glu Ala Val Ala Lys Ala Leu
260 265 270
Arg Pro Gly Gly Val Val Cys Thr Gln Ala Glu Ser Ile Trp Leu His
275 280 285
Met His Ile Ile Lys Gln Ile Ile Ala Asn Cys Arg Gln Val Phe Lys
290 295 300
Gly Ser Val Asn Tyr Ala Trp Thr Thr Val Pro Thr Tyr Pro Thr Gly
305 310 315 320
Val Ile Gly Tyr Met Leu Cys Ser Thr Glu Gly Pro Glu Val Asp Phe
325 330 335
Lys Asn Pro Val Asn Pro Ile Asp Lys Glu Thr Thr Gln Val Lys Ser
340 345 350
Lys Leu Ala Pro Leu Lys Phe Tyr Asn Ser Asp Ile His Lys Ala Ala
355 360 365
Phe Ile Leu Pro Ser Phe Ala Arg Ser Met Ile Glu Ser
370 375 380

<210> 15
<211> 419
<212> PRT
<213> Nicotiana tabacum
<400> 15
Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn
1 5 10 15
Gly Ala Ile Pro Met Asn Gly His Gln Ser Gly Thr Ser Lys His Leu
20 25 30
Asn Gly Tyr Gln Asn Gly Thr Ser Lys His Gln Asn Gly His His Asn
35 40 45
Gly Thr Ser Glu His Arg Asn Gly His Gln Asn Gly Ile Ser Glu His
50 55 60
Gln Asn Gly His Gln Asn Gly Thr Ser Glu His Arg Asn Gly His Gln
65 70 75 80
Asn Gly Ile Ser Glu His Gln Asn Gly His Gln Asn Gly Thr Ser Glu
85 90 95
His Gln Asn Gly His Gln Asn Gly Thr Ser Glu Gln Gln Asn Gly Thr
100 105 110
Ile Ser His Asp Asn Gly Asn Glu Leu Leu Gly Asn Ser Asn Ser Ile
115 120 125
Lys Leu Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp Pro Gly Glu Ala
130 135 140
Phe Ser Leu Lys Val Glu Lys Leu Leu Phe Gln Gly Lys Ser Asp Tyr
145 150 155 160
Gln Asp Val Met Leu Phe Glu Ser Ala Thr Tyr Gly Lys Val Leu Thr
165 170 175
Leu Asp Gly Ala Ile Gln His Thr Glu Asn Gly Gly Phe Pro Tyr Thr
180 185 190
Glu Met Ile Val His Leu Pro Leu Gly Ser Ile Pro Asn Pro Lys Lys
195 200 205
Val Leu Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu Phe Glu Met Leu
210 215 220
Arg Tyr Pro Thr Ile Glu Lys Ile Asp Ile Val Glu Ile Asp Asp Val
225 230 235 240
Val Val Asp Val Ser Arg Lys Ser Phe Pro Tyr Leu Ala Ala Asn Phe
245 250 255
Asn Asp Pro Arg Val Thr Leu Val Leu Gly Asp Gly Ala Ala Phe Val
260 265 270
Lys Ala Ala Gln Ala Gly Tyr Tyr Asp Ala Ile Ile Val Asp Ser Ser
275 280 285
Asp Pro Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg Pro Phe Phe Glu
290 295 300
Ala Val Ala Lys Ala Leu Arg Pro Gly Gly Val Val Cys Thr Gln Ala
305 310 315 320
Glu Ser Ile Trp Leu His Met His Ile Ile Lys Gln Ile Ile Ala Asn
325 330 335
Cys Arg Gln Val Phe Lys Gly Ser Val Asn Tyr Ala Trp Thr Thr Val
340 345 350
Pro Thr Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu Cys Ser Thr Glu
355 360 365
Gly Pro Glu Val Asp Phe Lys Asn Pro Ile Asn Pro Ile Asp Lys Glu
370 375 380
Thr Thr Gln Val Lys Ser Lys Leu Ala Pro Leu Lys Phe Tyr Asn Ser
385 390 395 400
Asp Ile His Lys Ala Ala Phe Ile Leu Pro Ser Phe Ala Arg Ser Met
405 410 415
Ile Glu Ser

<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 16
cccatgaacg gccaccaaaa 20

<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 17
ggcacttcca aacaccaaaa 20

<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 18
gttgttcgga tgtcccattc 20

<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 19
ctaaactctg aaaaccaacc 20

<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 20
tttcagagtt tagcgcatta 20

<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 21
gatggagcaa ttcaacatac aga 23

<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 22
gatggagcaa ttcaacacac aga 23

<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 23
tggcatttcc aaacaccaaa cgggcaccag aatggcactt 40

<210> 24
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 24
ccaactctat taagcctggt ggttttcaga gtttagcgca 40

<210> 25
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 25
ttctgacttt ggatggagca atacagagaa tggtggattt 40

<210> 26
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 26
ctgactttgg atggagcaat gagaatggtg gatttccata 40

<210> 27
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 27
tgactttgga tggagcaatt cagagaatgg tggatttcca 40

<210> 28
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 28
tgactttgga tggagcaatt agaatggtgg atttccatac 40

<210> 29
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 29
gactttggat ggagcaattc agagaatggt ggatttccat 40

<210> 30
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 30
gactttggat ggagcaattc gagaatggtg gatttccata 40

<210> 31
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 31
gactttggat ggagcaattc agaatggtgg atttccatac 40

<210> 32
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 32
gactttggat ggagcaattc tggatttcca tacactgaaa 40

<210> 33
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 33
actttggatg gagcaattca tacagagaat ggtggatttc c 41

<210> 34
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 34
actttggatg gagcaattca cagagaatgg tggatttcca 40

<210> 35
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 35
actttggatg gagcaattca agagaatggt ggatttccat 40

<210> 36
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 36
actttggatg gagcaattca gagaatggtg gatttccata 40

<210> 37
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 37
actttggatg gagcaattca atacactgaa atgattgttc 40

<210> 38
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 38
ctttggatgg agcaattcaa tacagagaat ggtggatttc 40

<210> 39
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 39
ctttggatgg agcaattcaa cagagaatgg tggatttcca 40

<210> 40
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 40
ctttggatgg agcaattcaa gagaatggtg gatttccata 40

<210> 41
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 41
ctttggatgg agcaattcaa agaatggtgg atttccatac 40

<210> 42
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 42
tttggatgga gcaattcaac tacagagaat ggtggatttc 40

<210> 43
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 43
tttggatgga gcaattcaac cagagaatgg tggatttcca 40

<210> 44
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 44
tttggatgga gcaattcaac agagaatggt ggatttccat 40

<210> 45
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 45
tttggatgga gcaattcaac gagaatggtg gatttccata 40

<210> 46
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 46
tttggatgga gcaattcaac agaatggtgg atttccatac 40

<210> 47
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 47
tttggatgga gcaattcaac aatggtggat ttccatacac 40

<210> 48
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 48
ttggatggag caattcaaca cagagaatgg tggatttcca 40

<210> 49
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 49
ttggatggag caattcaaca gagaatggtg gatttccata 40

<210> 50
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 50
tggatggagc aattcaacat agagaatggt ggatttccat 40

<210> 51
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 51
tggatggagc aattcaacat gagaatggtg gatttccata 40

<210> 52
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 52
tggatggagc aattcaacat agaatggtgg atttccatac 40

<210> 53
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 53
tggatggagc aattcaacat aatggtggat ttccatacac 40

<210> 54
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 54
ggatggagca attcaacata gagaatggtg gatttccata 40

<210> 55
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 55
caattcaaca tacagagaat gtggatttcc atacactgaa 40

<210> 56
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 56
gcacttccaa acaccaaaac agggcaccag aatggcactt t 41

<210> 57
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 57
ttctgacttt ggatggagca atacagagaa tggtggattt 40

<210> 58
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 58
ttctgacttt ggatggagca gagaatggtg gatttccata 40

<210> 59
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 59
ctgactttgg atggagcaat acagagaatg gtggatttcc 40

<210> 60
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 60
ctgactttgg atggagcaat gagaatggtg gatttccata 40

<210> 61
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 61
tgactttgga tggagcaatt cagagaatgg tggatttcca 40

<210> 62
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 62
tgactttgga tggagcaatt agaatggtgg atttccatac 40

<210> 63
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 63
gactttggat ggagcaattc agagaatggt ggatttccat 40

<210> 64
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 64
gactttggat ggagcaattc gagaatggtg gatttccata 40

<210> 65
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 65
gactttggat ggagcaattc agaatggtgg atttccatac 40

<210> 66
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 66
gactttggat ggagcaattc gaatggtgga tttccataca 40

<210> 67
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 67
gactttggat ggagcaattc tggatttcca tacactgaaa 40

<210> 68
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 68
actttggatg gagcaattca atacagagaa tggtggattt 40

<210> 69
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 69
actttggatg gagcaattca acagagaatg gtggatttcc 40

<210> 70
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 70
actttggatg gagcaattca agagaatggt ggatttccat 40

<210> 71
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 71
actttggatg gagcaattca gagaatggtg gatttccata 40

<210> 72
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 72
actttggatg gagcaattca agaatggtgg atttccatac 40

<210> 73
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 73
actttggatg gagcaattca aatggtggat ttccatacac 40

<210> 74
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 74
ctttggatgg agcaattcaa atacagagaa tggtggattt 40

<210> 75
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 75
ctttggatgg agcaattcaa tacagagaat ggtggatttc 40

<210> 76
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 76
ctttggatgg agcaattcaa cagagaatgg tggatttcca 40

<210> 77
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 77
ctttggatgg agcaattcaa agagaatggt ggatttccat 40

<210> 78
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 78
ctttggatgg agcaattcaa agaatggtgg atttccatac 40

<210> 79
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 79
tttggatgga gcaattcaac tacagagaat ggtggatttc c 41

<210> 80
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 80
tttggatgga gcaattcaac agagaatggt ggatttccat 40

<210> 81
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 81
tttggatgga gcaattcaac gagaatggtg gatttccata 40

<210> 82
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 82
tttggatgga gcaattcaac agaatggtgg atttccatac 40

<210> 83
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 83
tttggatgga gcaattcaac gaatggtgga tttccataca 40

<210> 84
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 84
tttggatgga gcaattcaac ggatttccat acactgaaat 40

<210> 85
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 85
ttggatggag caattcaaca acagagaatg gtggatttcc 40

<210> 86
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 86
ttggatggag caattcaaca cagagaatgg tggatttcca 40

<210> 87
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 87
ttggatggag caattcaaca agagaatggt ggatttccat 40

<210> 88
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 88
ttggatggag caattcaaca gagaatggtg gatttccata 40

<210> 89
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 89
ttggatggag caattcaaca agaatggtgg atttccatac 40

<210> 90
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 90
ttggatggag caattcaaca gaatggtgga tttccataca 40

<210> 91
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 91
ttggatggag caattcaaca aatggtggat ttccatacac 40

<210> 92
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 92
tggatggagc aattcaacat agagaatggt ggatttccat 40

<210> 93
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 93
tggatggagc aattcaacat agaatggtgg atttccatac 40

<210> 94
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 94
tggatggagc aattcaacat atggtggatt tccatacact 40

<210> 95
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 95
ggatggagca attcaacata gagaatggtg gatttccata 40

<210> 96
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 96
ggatggagca attcaacata agaatggtgg atttccatac 40

<210> 97
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 97
gagcaattca acatacagag tggtggattt ccatacactg 40

<210> 98
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 98
tggcacttcc aaacaccaaa cggccacaag aatgggactt 40

<210> 99
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 99
ccaattgtat taagcctggt ggttttcaga gtttagcgca 40

<210> 100
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 100
tgactttgga tggagcaatt cacagagaat ggtggatttc 40

<210> 101
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 101
tgactttgga tggagcaatt cagagaatgg tggatttcca 40

<210> 102
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 102
tgactttgga tggagcaatt agaatggtgg atttccatac 40

<210> 103
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 103
gactttggat ggagcaattc gagaatggtg gatttccata 40

<210> 104
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 104
gactttggat ggagcaattc aatggtggat ttccatacac 40

<210> 105
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 105
actttggatg gagcaattca acacagagaa tggtggattt 40

<210> 106
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 106
actttggatg gagcaattca acagagaatg gtggatttcc 40

<210> 107
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 107
actttggatg gagcaattca cagagaatgg tggatttcca 40

<210> 108
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 108
actttggatg gagcaattca agagaatggt ggatttccat 40

<210> 109
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 109
actttggatg gagcaattca gagaatggtg gatttccata 40

<210> 110
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 110
actttggatg gagcaattca agaatggtgg atttccatac 40

<210> 111
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 111
actttggatg gagcaattca aatggtggat ttccatacac 40

<210> 112
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 112
ctttggatgg agcaattcaa acacagagaa tggtggattt 40

<210> 113
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 113
ctttggatgg agcaattcaa cacagagaat ggtggatttc 40

<210> 114
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 114
ctttggatgg agcaattcaa cagagaatgg tggatttcca 40

<210> 115
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 115
ctttggatgg agcaattcaa agaatggtgg atttccatac 40

<210> 116
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 116
tttggatgga gcaattcaac acagagaatg gtggatttcc 40

<210> 117
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 117
tttggatgga gcaattcaac agagaatggt ggatttccat 40

<210> 118
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 118
tttggatgga gcaattcaac gagaatggtg gatttccata 40

<210> 119
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 119
tttggatgga gcaattcaac agaatggtgg atttccatac 40

<210> 120
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 120
tttggatgga gcaattcaac aatggtggat ttccatacac 40

<210> 121
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 121
tttggatgga gcaattcaac ggatttccat acactgaaat 40

<210> 122
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 122
ttggatggag caattcaaca cagagaatgg tggatttcca 40

<210> 123
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 123
ttggatggag caattcaaca gagaatggtg gatttccata 40

<210> 124
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 124
ttggatggag caattcaaca gaatggtgga tttccataca 40

<210> 125
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 125
ttggatggag caattcaaca aatggtggat ttccatacac 40

<210> 126
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 126
ttggatggag caattcaaca tggatttcca tacactgaaa 40

<210> 127
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 127
tggatggagc aattcaacac gagaatggtg gatttccata 40

<210> 128
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 128
tggatggagc aattcaacac agaatggtgg atttccatac 40

<210> 129
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 129
ggatggagca attcaacaca gagaatggtg gatttccata 40

<210> 130
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 130
ggatggagca attcaacaca gaatggtgga tttccataca 40

<210> 131
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 131
ggatggagca attcaacaca gtggatttcc atacactgaa 40

<210> 132
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 132
gatggagcaa ttcaacacac tggtggattt ccatacactg 40

<210> 133
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 133
tggcacttcc aaacaccaaa cggccaccag aatggcactt 40

<210> 134
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 134
ccaactctat taagcctggt ggttttcaga gtttagcgca 40

<210> 135
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 135
aacatatggg aaggttctga ttggatggag caattcaaca 40

<210> 136
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 136
atgggaaggt tctgactttg tggagcaatt caacacacag 40

<210> 137
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 137
gttctgactt tggatggagc ttcaacacac agagaatggt 40

<210> 138
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 138
ctgactttgg atggagcaat acagagaatg gtggatttcc 40

<210> 139
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 139
ctgactttgg atggagcaat gagaatggtg gatttccata 40

<210> 140
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 140
ctgactttgg atggagcaat agaatggtgg atttccatac 40

<210> 141
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 141
tgactttgga tggagcaatt cagagaatgg tggatttcca 40

<210> 142
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 142
gactttggat ggagcaattc acacagag aatggtggat 40

<210> 143
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 143
gactttggat ggagcaattc cacacagaga atggtggatt 40

<210> 144
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 144
gactttggat ggagcaattc gagaatggtg gatttccata 40

<210> 145
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 145
gactttggat ggagcaattc aatggtggat ttccatacac 40

<210> 146
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 146
actttggatg gagcaattca acacagagaa tggtggattt 40

<210> 147
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 147
actttggatg gagcaattca acagagaatg gtggatttcc 40

<210> 148
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 148
actttggatg gagcaattca agagaatggt ggatttccat 40

<210> 149
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 149
actttggatg gagcaattca agaatggtgg atttccatac 40

<210> 150
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 150
actttggatg gagcaattca aatggtggat ttccatacac 40

<210> 151
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 151
actttggatg gagcaattca tggatttcca tacactgaaa 40

<210> 152
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 152
actttggatg gagcaattca gatttccata cactgaaatg 40

<210> 153
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 153
ctttggatgg agcaattcaa acacagagaa tggtggattt 40

<210> 154
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 154
ctttggatgg agcaattcaa cagagaatgg tggatttcca 40

<210> 155
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 155
ctttggatgg agcaattcaa agagaatggt ggatttccat 40

<210> 156
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 156
ctttggatgg agcaattcaa agaatggtgg atttccatac 40

<210> 157
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 157
tttggatgga gcaattcaac acagagaatg gtggatttcc 40

<210> 158
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 158
tttggatgga gcaattcaac agagaatggt ggatttccat 40

<210> 159
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 159
tttggatgga gcaattcaac gagaatggtg gatttccata 40

<210> 160
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 160
ttggatggag caattcaaca cagagaatgg tggatttcca 40

<210> 161
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 161
ttggatggag caattcaaca gagaatggtg gatttccata 40

<210> 162
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 162
ttggatggag caattcaaca agaatggtgg atttccatac 40

<210> 163
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 163
ttggatggag caattcaaca ggatttccat acactgaaat 40

<210> 164
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 164
tggatggagc aattcaacac agagaatggt ggatttccat 40

<210> 165
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 165
tggatggagc aattcaacac agaatggtgg atttccatac 40

<210> 166
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 166
tggatggagc aattcaacac tggtggattt ccatacactg 40

<210> 167
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 167
ggatggagca attcaacaca gagaatggtg gatttccata 40

<210> 168
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 168
tggagcaatt caacacacag atggtggatt tccatacact 40

<210> 169
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 169
tggagcaatt caacacacag tggtggattt ccatacactg 40

<210> 170
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 170
ggagcaattc aacacacaga aatggtggat ttccatacac 40

<210> 171
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 171
caattcaaca cacagagaat atttccatac actgaaatga 40

<210> 172
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 172
aacacacaga gaatggtgga aaatgattgt tcatcttcca 40

<210> 173
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 173
cggcacttcc aaacaccaaa cggccaccat aatggcactt 40

<210> 174
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 174
ttttgacttt ggatggagca agagaatggt ggatttccat 40

<210> 175
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 175
ttgactttgg atggagcaat gaatggtgga tttccataca 40

<210> 176
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 176
tgactttgga tggagcaatt cagagaatgg tggatttcca 40

<210> 177
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 177
tgactttgga tggagcaatt agaatggtgg atttccatac 40

<210> 178
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 178
gactttggat ggagcaattc aatggtggat ttccatacac 40

<210> 179
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 179
gactttggat ggagcaattc tggtggattt ccatacactg 40

<210> 180
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 180
actttggatg gagcaattca acacagagaa tggtggattt 40

<210> 181
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 181
actttggatg gagcaattca acagagaatg gtggatttcc 40

<210> 182
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 182
actttggatg gagcaattca agagaatggt ggatttccat 40

<210> 183
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 183
actttggatg gagcaattca gagaatggtg gatttccata 40

<210> 184
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 184
actttggatg gagcaattca agaatggtgg atttccatac 40

<210> 185
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 185
ctttggatgg agcaattcaa cagagaatgg tggatttcca 40

<210> 186
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 186
ctttggatgg agcaattcaa agagaatggt ggatttccat 40

<210> 187
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 187
tttggatgga gcaattcaac acagagaatg gtggatttcc 40

<210> 188
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 188
tttggatgga gcaattcaac agagaatggt ggatttccat 40

<210> 189
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 189
tttggatgga gcaattcaac gagaatggtg gatttccata 40

<210> 190
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 190
ttggatggag caattcaaca gagaatggtg gatttccata 40

<210> 191
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 191
ttggatggag caattcaaca agaatggtgg atttccatac 40

<210> 192
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 192
tggatggagc aattcaacac agagaatggt ggatttccat 40

<210> 193
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 193
tggatggagc aattcaacac gagaatggtg gatttccata 40

<210> 194
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 194
tggatggagc aattcaacac agaatggtgg atttccatac 40

<210> 195
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 195
tggatggagc aattcaacac atggtggatt tccatacact 40

<210> 196
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 196
ggatggagca attcaacaca gagaatggtg gatttccata 40

<210> 197
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 197
tggagcaatt caacacacag gaatggtgga tttccataca 40

<210> 198
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 198
gcaattcaac acacagagaa atttccatac actgaaatga 40

<210> 199
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 199
aacacacaga gaatggtgga tccatacact gaaatgattg 40

<210> 200
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 200
cacagagaat ggtggatttc cactgaaatg attgttcatc 40

<210> 201
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 201
tggcatttcc aaacaccaaa acgggcacca gaatggcact t 41

<210> 202
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 202
ccaactctat taagcctggt tggttttcag agtttagcgc a 41

<210> 203
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 203
ttctgacttt ggatggagca attcaacata cagagaatgg tggattt 47

<210> 204
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 204
ctgactttgg atggagcaat tcaacataca gagaatggtg gatttccata 50

<210> 205
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 205
tgactttgga tggagcaatt caacatacag agaatggtgg atttcca 47

<210> 206
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 206
tgactttgga tggagcaatt caacatacag agaatggtgg atttccatac 50

<210> 207
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 207
gactttggat ggagcaattc aacatacaga gaatggtgga tttccat 47

<210> 208
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 208
gactttggat ggagcaattc aacatacaga gaatggtgga tttccata 48

<210> 209
<211> 49
<212> DNA
<213> Nicotiana tabacum
<400> 209
gactttggat ggagcaattc aacatacaga gaatggtgga tttccatac 49

<210> 210
<211> 56
<212> DNA
<213> Nicotiana tabacum
<400> 210
gactttggat ggagcaattc aacatacaga gaatggtgga tttccataca ctgaaa 56

<210> 211
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 211
actttggatg gagcaattca acatacagag aatggtggat ttcc 44

<210> 212
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 212
actttggatg gagcaattca acatacagag aatggtggat ttcca 45

<210> 213
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 213
actttggatg gagcaattca acatacagag aatggtggat ttccat 46

<210> 214
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 214
actttggatg gagcaattca acatacagag aatggtggat ttccata 47

<210> 215
<211> 64
<212> DNA
<213> Nicotiana tabacum
<400> 215
actttggatg gagcaattca acatacagag aatggtggat ttccatacac tgaaatgatt 60
gttc 64

<210> 216
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 216
ctttggatgg agcaattcaa catacagaga atggtggatt tc 42

<210> 217
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 217
ctttggatgg agcaattcaa catacagaga atggtggatt tcca 44

<210> 218
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 218
ctttggatgg agcaattcaa catacagaga atggtggatt tccata 46

<210> 219
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 219
ctttggatgg agcaattcaa catacagaga atggtggatt tccatac 47

<210> 220
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 220
tttggatgga gcaattcaac atacagagaa tggtggattt c 41

<210> 221
<211> 43
<212> DNA
<213> Nicotiana tabacum
<400> 221
tttggatgga gcaattcaac atacagagaa tggtggattt cca 43

<210> 222
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 222
tttggatgga gcaattcaac atacagagaa tggtggattt ccat 44

<210> 223
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 223
tttggatgga gcaattcaac atacagagaa tggtggattt ccata 45

<210> 224
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 224
tttggatgga gcaattcaac atacagagaa tggtggattt ccatac 46

<210> 225
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 225
tttggatgga gcaattcaac atacagagaa tggtggattt ccatacac 48

<210> 226
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 226
ttggatggag caattcaaca tacagagaat ggtggatttc ca 42

<210> 227
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 227
ttggatggag caattcaaca tacagagaat ggtggatttc cata 44

<210> 228
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 228
tggatggagc aattcaacat acagagaatg gtggatttcc at 42

<210> 229
<211> 43
<212> DNA
<213> Nicotiana tabacum
<400> 229
tggatggagc aattcaacat acagagaatg gtggatttcc ata 43

<210> 230
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 230
tggatggagc aattcaacat acagagaatg gtggatttcc atac 44

<210> 231
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 231
tggatggagc aattcaacat acagagaatg gtggatttcc atacac 46

<210> 232
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 232
ggatggagca attcaacata cagagaatgg tggatttcca ta 42

<210> 233
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 233
caattcaaca tacagagaat ggtggatttc catacactga a 41

<210> 234
<211> 40
<212> DNA
<213> Nicotiana tabacum
<400> 234
gcacttccaa acaccaaaac gggcaccaga atggcacttt 40

<210> 235
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 235
ttctgacttt ggatggagca attcaacata cagagaatgg tggattt 47

<210> 236
<211> 52
<212> DNA
<213> Nicotiana tabacum
<400> 236
ttctgacttt ggatggagca attcaacata cagagaatgg tggatttcca ta 52

<210> 237
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 237
ctgactttgg atggagcaat tcaacataca gagaatggtg gatttcc 47

<210> 238
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 238
ctgactttgg atggagcaat tcaacataca gagaatggtg gatttccata 50

<210> 239
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 239
tgactttgga tggagcaatt caacatacag agaatggtgg atttcca 47

<210> 240
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 240
tgactttgga tggagcaatt caacatacag agaatggtgg atttccatac 50

<210> 241
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 241
gactttggat ggagcaattc aacatacaga gaatggtgga tttccat 47

<210> 242
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 242
gactttggat ggagcaattc aacatacaga gaatggtgga tttccata 48

<210> 243
<211> 49
<212> DNA
<213> Nicotiana tabacum
<400> 243
gactttggat ggagcaattc aacatacaga gaatggtgga tttccatac 49

<210> 244
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 244
gactttggat ggagcaattc aacatacaga gaatggtgga tttccataca 50

<210> 245
<211> 56
<212> DNA
<213> Nicotiana tabacum
<400> 245
gactttggat ggagcaattc aacatacaga gaatggtgga tttccataca ctgaaa 56

<210> 246
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 246
actttggatg gagcaattca acatacagag aatggtggat tt 42

<210> 247
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 247
actttggatg gagcaattca acatacagag aatggtggat ttcc 44

<210> 248
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 248
actttggatg gagcaattca acatacagag aatggtggat ttccat 46

<210> 249
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 249
actttggatg gagcaattca acatacagag aatggtggat ttccata 47

<210> 250
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 250
actttggatg gagcaattca acatacagag aatggtggat ttccatac 48

<210> 251
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 251
actttggatg gagcaattca acatacagag aatggtggat ttccatacac 50

<210> 252
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 252
ctttggatgg agcaattcaa catacagaga atggtggatt t 41

<210> 253
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 253
ctttggatgg agcaattcaa catacagaga atggtggatt tc 42

<210> 254
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 254
ctttggatgg agcaattcaa catacagaga atggtggatt tcca 44

<210> 255
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 255
ctttggatgg agcaattcaa catacagaga atggtggatt tccat 45

<210> 256
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 256
ctttggatgg agcaattcaa catacagaga atggtggatt tccatac 47

<210> 257
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 257
tttggatgga gcaattcaac atacagagaa tggtggattt cc 42

<210> 258
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 258
tttggatgga gcaattcaac atacagagaa tggtggattt ccat 44

<210> 259
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 259
tttggatgga gcaattcaac atacagagaa tggtggattt ccata 45

<210> 260
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 260
tttggatgga gcaattcaac atacagagaa tggtggattt ccatac 46

<210> 261
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 261
tttggatgga gcaattcaac atacagagaa tggtggattt ccataca 47

<210> 262
<211> 54
<212> DNA
<213> Nicotiana tabacum
<400> 262
tttggatgga gcaattcaac atacagagaa tggtggattt ccatacactg aaat 54

<210> 263
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 263
ttggatggag caattcaaca tacagagaat ggtggatttc c 41

<210> 264
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 264
ttggatggag caattcaaca tacagagaat ggtggatttc ca 42

<210> 265
<211> 43
<212> DNA
<213> Nicotiana tabacum
<400> 265
ttggatggag caattcaaca tacagagaat ggtggatttc cat 43

<210> 266
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 266
ttggatggag caattcaaca tacagagaat ggtggatttc cata 44

<210> 267
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 267
ttggatggag caattcaaca tacagagaat ggtggatttc catac 45

<210> 268
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 268
ttggatggag caattcaaca tacagagaat ggtggatttc cataca 46

<210> 269
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 269
ttggatggag caattcaaca tacagagaat ggtggatttc catacac 47

<210> 270
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 270
tggatggagc aattcaacat acagagaatg gtggatttcc at 42

<210> 271
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 271
tggatggagc aattcaacat acagagaatg gtggatttcc atac 44

<210> 272
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 272
tggatggagc aattcaacat acagagaatg gtggatttcc atacact 47

<210> 273
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 273
ggatggagca attcaacata cagagaatgg tggatttcca ta 42

<210> 274
<211> 43
<212> DNA
<213> Nicotiana tabacum
<400> 274
ggatggagca attcaacata cagagaatgg tggatttcca tac 43

<210> 275
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 275
gagcaattca acatacagag aatggtggat ttccatacac tg 42

<210> 276
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 276
tggcacttcc aaacaccaaa acggccacaa gaatgggact t 41

<210> 277
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 277
ccaattgtat taagcctggt tggttttcag agtttagcgc a 41

<210> 278
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 278
tgactttgga tggagcaatt caacacacag agaatggtgg atttc 45

<210> 279
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 279
tgactttgga tggagcaatt caacacacag agaatggtgg atttcca 47

<210> 280
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 280
tgactttgga tggagcaatt caacacacag agaatggtgg atttccatac 50

<210> 281
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 281
gactttggat ggagcaattc aacacacaga gaatggtgga tttccata 48

<210> 282
<211> 51
<212> DNA
<213> Nicotiana tabacum
<400> 282
gactttggat ggagcaattc aacacacaga gaatggtgga tttccataca c 51

<210> 283
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 283
actttggatg gagcaattca aacacacagag aatggtggat tt 42

<210> 284
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 284
actttggatg gagcaattca aacacacagag aatggtggat ttcc 44

<210> 285
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 285
actttggatg gagcaattca aacacacagag aatggtggat ttcca 45

<210> 286
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 286
actttggatg gagcaattca aacacacagag aatggtggat ttccat 46

<210> 287
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 287
actttggatg gagcaattca aacacacagag aatggtggat ttccata 47

<210> 288
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 288
actttggatg gagcaattca aacacacagag aatggtggat ttccatac 48

<210> 289
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 289
actttggatg gagcaattca aacacacagag aatggtggat ttccatacac 50

<210> 290
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 290
ctttggatgg agcaattcaa cacacagaga atggtggatt t 41

<210> 291
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 291
ctttggatgg agcaattcaa cacacagaga atggtggatt tc 42

<210> 292
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 292
ctttggatgg agcaattcaa cacacagaga atggtggatt tcca 44

<210> 293
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 293
ctttggatgg agcaattcaa cacacagaga atggtggatt tccatac 47

<210> 294
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 294
tttggatgga gcaattcaac acacagagaa tggtggattt cc 42

<210> 295
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 295
tttggatgga gcaattcaac acacagagaa tggtggattt ccat 44

<210> 296
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 296
tttggatgga gcaattcaac acacagagaa tggtggattt ccata 45

<210> 297
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 297
tttggatgga gcaattcaac acacagagaa tggtggattt ccatac 46

<210> 298
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 298
tttggatgga gcaattcaac acacagagaa tggtggattt ccatacac 48

<210> 299
<211> 54
<212> DNA
<213> Nicotiana tabacum
<400> 299
tttggatgga gcaattcaac acacagagaa tggtggattt ccatacactg aaat 54

<210> 300
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 300
ttggatggag caattcaaca cacagagaat ggtggatttc ca 42

<210> 301
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 301
ttggatggag caattcaaca cacagagaat ggtggatttc cata 44

<210> 302
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 302
ttggatggag caattcaaca cacagagaat ggtggatttc cataca 46

<210> 303
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 303
ttggatggag caattcaaca cacagagaat ggtggatttc catacac 47

<210> 304
<211> 52
<212> DNA
<213> Nicotiana tabacum
<400> 304
ttggatggag caattcaaca cacagagaat ggtggatttc catacactga aa 52

<210> 305
<211> 43
<212> DNA
<213> Nicotiana tabacum
<400> 305
tggatggagc aattcaacac acagagaatg gtggatttcc ata 43

<210> 306
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 306
tggatggagc aattcaacac acagagaatg gtggatttcc atac 44

<210> 307
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 307
ggatggagca attcaacaca cagagaatgg tggatttcca ta 42

<210> 308
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 308
ggatggagca attcaacaca cagagaatgg tggatttcca taca 44

<210> 309
<211> 49
<212> DNA
<213> Nicotiana tabacum
<400> 309
ggatggagca attcaacaca cagagaatgg tggatttcca tacactgaa 49

<210> 310
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 310
gatggagcaa ttcaacacac agagaatggt ggatttccat acactg 46

<210> 311
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 311
tggcacttcc aaacaccaaa acggccacca gaatggcact t 41

<210> 312
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 312
ccaactctat taagcctggt tggttttcag agtttagcgc a 41

<210> 313
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 313
aacatatggg aaggttctga ctttggatgg agcaattcaa ca 42

<210> 314
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 314
atgggaaggt tctgactttg gatggagcaa ttcaacacac ag 42

<210> 315
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 315
gttctgactt tggatggagc aattcaacac acagagaatg gt 42

<210> 316
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 316
ctgactttgg atggagcaat tcaacacaca gagaatggtg gatttcc 47

<210> 317
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 317
ctgactttgg atggagcaat tcaacacaca gagaatggtg gatttccata 50

<210> 318
<211> 51
<212> DNA
<213> Nicotiana tabacum
<400> 318
ctgactttgg atggagcaat tcaacacaca gagaatggtg gatttccata c 51

<210> 319
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 319
tgactttgga tggagcaatt caacacacag agaatggtgg atttcca 47

<210> 320
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 320
gactttggat ggagcaattc aacacacaga gaatggtgga t 41

<210> 321
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 321
gactttggat ggagcaattc aacacacaga gaatggtgga tt 42

<210> 322
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 322
gactttggat ggagcaattc aacacacaga gaatggtgga tttccata 48

<210> 323
<211> 51
<212> DNA
<213> Nicotiana tabacum
<400> 323
gactttggat ggagcaattc aacacacaga gaatggtgga tttccataca c 51

<210> 324
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 324
actttggatg gagcaattca aacacacagag aatggtggat tt 42

<210> 325
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 325
actttggatg gagcaattca aacacacagag aatggtggat ttcc 44

<210> 326
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 326
actttggatg gagcaattca aacacacagag aatggtggat ttccat 46

<210> 327
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 327
actttggatg gagcaattca aacacacagag aatggtggat ttccatac 48

<210> 328
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 328
actttggatg gagcaattca aacacacagag aatggtggat ttccatacac 50

<210> 329
<211> 55
<212> DNA
<213> Nicotiana tabacum
<400> 329
actttggatg gagcaattca aacacacagag aatggtggat ttccatacac tgaaa 55

<210> 330
<211> 57
<212> DNA
<213> Nicotiana tabacum
<400> 330
actttggatg gagcaattca aacacacagag aatggtggat ttccatacac tgaaatg 57

<210> 331
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 331
ctttggatgg agcaattcaa cacacagaga atggtggatt t 41

<210> 332
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 332
ctttggatgg agcaattcaa cacacagaga atggtggatt tcca 44

<210> 333
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 333
ctttggatgg agcaattcaa cacacagaga atggtggatt tccat 45

<210> 334
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 334
ctttggatgg agcaattcaa cacacagaga atggtggatt tccatac 47

<210> 335
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 335
tttggatgga gcaattcaac acacagagaa tggtggattt cc 42

<210> 336
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 336
tttggatgga gcaattcaac acacagagaa tggtggattt ccat 44

<210> 337
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 337
tttggatgga gcaattcaac acacagagaa tggtggattt ccata 45

<210> 338
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 338
ttggatggag caattcaaca cacagagaat ggtggatttc ca 42

<210> 339
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 339
ttggatggag caattcaaca cacagagaat ggtggatttc cata 44

<210> 340
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 340
ttggatggag caattcaaca cacagagaat ggtggatttc catac 45

<210> 341
<211> 53
<212> DNA
<213> Nicotiana tabacum
<400> 341
ttggatggag caattcaaca cacagagaat ggtggatttc catacactga aat 53

<210> 342
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 342
tggatggagc aattcaacac acagagaatg gtggatttcc at 42

<210> 343
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 343
tggatggagc aattcaacac acagagaatg gtggatttcc atac 44

<210> 344
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 344
tggatggagc aattcaacac acagagaatg gtggatttcc atacactg 48

<210> 345
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 345
ggatggagca attcaacaca cagagaatgg tggatttcca ta 42

<210> 346
<211> 43
<212> DNA
<213> Nicotiana tabacum
<400> 346
tggagcaatt caacacacag agaatggtgg atttccatac act 43

<210> 347
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 347
tggagcaatt caacacacag agaatggtgg atttccatac actg 44

<210> 348
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 348
ggagcaattc aacacacaga gaatggtgga tttccataca c 41

<210> 349
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 349
caattcaaca cacagagaat ggtggatttc catacactga aatga 45

<210> 350
<211> 53
<212> DNA
<213> Nicotiana tabacum
<400> 350
aacacacaga gaatggtgga tttccataca ctgaaatgat tgttcatctt cca 53

<210> 351
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 351
cggcacttcc aaacaccaaa acggccacca taatggcact t 41

<210> 352
<211> 51
<212> DNA
<213> Nicotiana tabacum
<400> 352
ttttgacttt ggatggagca attcaacaca cagagaatgg tggatttcca t 51

<210> 353
<211> 52
<212> DNA
<213> Nicotiana tabacum
<400> 353
ttgactttgg atggagcaat tcaacacaca gagaatggtg gatttccata ca 52

<210> 354
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 354
tgactttgga tggagcaatt caacacacag agaatggtgg atttcca 47

<210> 355
<211> 50
<212> DNA
<213> Nicotiana tabacum
<400> 355
tgactttgga tggagcaatt caacacacag agaatggtgg atttccatac 50

<210> 356
<211> 51
<212> DNA
<213> Nicotiana tabacum
<400> 356
gactttggat ggagcaattc aacacacaga gaatggtgga tttccataca c 51

<210> 357
<211> 53
<212> DNA
<213> Nicotiana tabacum
<400> 357
gactttggat ggagcaattc aacacacaga gaatggtgga tttccataca ctg 53

<210> 358
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 358
actttggatg gagcaattca aacacacagag aatggtggat tt 42

<210> 359
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 359
actttggatg gagcaattca aacacacagag aatggtggat ttcc 44

<210> 360
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 360
actttggatg gagcaattca aacacacagag aatggtggat ttccat 46

<210> 361
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 361
actttggatg gagcaattca aacacacagag aatggtggat ttccata 47

<210> 362
<211> 48
<212> DNA
<213> Nicotiana tabacum
<400> 362
actttggatg gagcaattca aacacacagag aatggtggat ttccatac 48

<210> 363
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 363
ctttggatgg agcaattcaa cacacagaga atggtggatt tcca 44

<210> 364
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 364
ctttggatgg agcaattcaa cacacagaga atggtggatt tccat 45

<210> 365
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 365
tttggatgga gcaattcaac acacagagaa tggtggattt cc 42

<210> 366
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 366
tttggatgga gcaattcaac acacagagaa tggtggattt ccat 44

<210> 367
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 367
tttggatgga gcaattcaac acacagagaa tggtggattt ccata 45

<210> 368
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 368
ttggatggag caattcaaca cacagagaat ggtggatttc cata 44

<210> 369
<211> 45
<212> DNA
<213> Nicotiana tabacum
<400> 369
ttggatggag caattcaaca cacagagaat ggtggatttc catac 45

<210> 370
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 370
tggatggagc aattcaacac acagagaatg gtggatttcc at 42

<210> 371
<211> 43
<212> DNA
<213> Nicotiana tabacum
<400> 371
tggatggagc aattcaacac acagagaatg gtggatttcc ata 43

<210> 372
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 372
tggatggagc aattcaacac acagagaatg gtggatttcc atac 44

<210> 373
<211> 47
<212> DNA
<213> Nicotiana tabacum
<400> 373
tggatggagc aattcaacac acagagaatg gtggatttcc atacact 47

<210> 374
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 374
ggatggagca attcaacaca cagagaatgg tggatttcca ta 42

<210> 375
<211> 41
<212> DNA
<213> Nicotiana tabacum
<400> 375
tggagcaatt caacacacag agaatggtgg atttccatac a 41

<210> 376
<211> 46
<212> DNA
<213> Nicotiana tabacum
<400> 376
gcaattcaac acacagagaa tggtggattt ccatacactg aaatga 46

<210> 377
<211> 42
<212> DNA
<213> Nicotiana tabacum
<400> 377
aacacacaga gaatggtgga tttccataca ctgaaatgat tg 42

<210> 378
<211> 44
<212> DNA
<213> Nicotiana tabacum
<400> 378
cacagagaat ggtggatttc catacactga aatgattgtt catc 44

<210> 379
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 379
tcaacataca 10

<210> 380
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 380
caacatacag 10

<210> 381
<211> 16
<212> DNA
<213> Nicotiana tabacum
<400> 381
aacatacaga gaatgg 16

<210> 382
<211> 24
<212> DNA
<213> Nicotiana tabacum
<400> 382
acatacagag aatggtggat ttcc 24

<210> 383
<211> 12
<212> DNA
<213> Nicotiana tabacum
<400> 383
Attcaacata ca 12

<210> 384
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 384
tcaacataca 10

<210> 385
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 385
caacatacag 10

<210> 386
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 386
aacatacaga 10

<210> 387
<211> 16
<212> DNA
<213> Nicotiana tabacum
<400> 387
aacatacaga gaatgg 16

<210> 388
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 388
acatacagag 10

<210> 389
<211> 14
<212> DNA
<213> Nicotiana tabacum
<400> 389
atacagagaa tggt 14

<210> 390
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 390
caacacacag 10

<210> 391
<211> 11
<212> DNA
<213> Nicotiana tabacum
<400> 391
aacacacaga g 11

<210> 392
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 392
acacacagag 10

<210> 393
<211> 14
<212> DNA
<213> Nicotiana tabacum
<400> 393
acacagagaa tggt 14

<210> 394
<211> 12
<212> DNA
<213> Nicotiana tabacum
<400> 394
cacagagaat gg 12

<210> 395
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 395
tcaacacaca 10

<210> 396
<211> 11
<212> DNA
<213> Nicotiana tabacum
<400> 396
tcaacacaca g 11

<210> 397
<211> 11
<212> DNA
<213> Nicotiana tabacum
<400> 397
aacacacaga g 11

<210> 398
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 398
acacacagag 10

<210> 399
<211> 15
<212> DNA
<213> Nicotiana tabacum
<400> 399
acacacagag aatgg 15

<210> 400
<211> 17
<212> DNA
<213> Nicotiana tabacum
<400> 400
acacacagag aatggtg 17

<210> 401
<211> 13
<212> DNA
<213> Nicotiana tabacum
<400> 401
cacagagaat ggt 13

<210> 402
<211> 13
<212> DNA
<213> Nicotiana tabacum
<400> 402
tttccataca ctg 13

<210> 403
<211> 11
<212> DNA
<213> Nicotiana tabacum
<400> 403
attcaacaca c 11

<210> 404
<211> 12
<212> DNA
<213> Nicotiana tabacum
<400> 404
tcaacacaca ga 12

<210> 405
<211> 10
<212> DNA
<213> Nicotiana tabacum
<400> 405
caacacacag 10

<210> 406
<211> 11
<212> DNA
<213> Nicotiana tabacum
<400> 406
aacacacaga g 11

<210> 407
<211> 13
<212> DNA
<213> Nicotiana tabacum
<400> 407
aacacacaga gaa 13

<210> 408
<211> 19
<212> DNA
<213> Nicotiana tabacum
<400> 408
gagcaattca acatacaga 19

<210> 409
<211> 13
<212> DNA
<213> Nicotiana tabacum
<400> 409
ggagcaattc aac 13

<210> 410
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 410
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 411
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 411
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 412
<211> 83
<212> DNA
<213> Nicotiana tabacum
<400> 412
tcagcaactt atgggaaggt tctgactttg gatggagcaa tgagaatggt ggatttccat 60
acactgaaat gattgttcat cta 83

<210> 413
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 413
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 414
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 414
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 415
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 415
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaaagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 416
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 416
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaaagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 417
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 417
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 418
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 418
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 419
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 419
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 420
<211> 74
<212> DNA
<213> Nicotiana tabacum
<400> 420
tcagcaactt atgggaaggt tctgactttg gatggaatgg tggatttcca tacactgaaa 60
tgattgttca tcta 74

<210> 421
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 421
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 422
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 422
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 423
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 423
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 424
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 424
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 425
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 425
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 426
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 426
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 427
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 427
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 428
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 428
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 429
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 429
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 430
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 430
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacaaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 431
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 431
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 432
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 432
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 433
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 433
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 434
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 434
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 435
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 435
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 436
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 436
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 437
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 437
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 438
<211> 80
<212> DNA
<213> Nicotiana tabacum
<400> 438
tcagcaactt atgggaaggt tctgactttg gatatacaga gaatggtgga tttccataca 60
ctgaaatgat tgttcatcta 80

<210> 439
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 439
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 440
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 440
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaatacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 441
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 441
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 442
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 442
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 443
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 443
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 444
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 444
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 445
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 445
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 446
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 446
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 447
<211> 104
<212> DNA
<213> Nicotiana tabacum
<400> 447
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctatcaaaga atgg 104

<210> 448
<211> 104
<212> DNA
<213> Nicotiana tabacum
<400> 448
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctatcaaaga atgg 104

<210> 449
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 449
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 450
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 450
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 451
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 451
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 452
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 452
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 453
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 453
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 454
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 454
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 455
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 455
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 456
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 456
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 457
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 457
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 458
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 458
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 459
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 459
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 460
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 460
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 461
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 461
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 462
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 462
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 463
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 463
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 464
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 464
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 465
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 465
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 466
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 466
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 467
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 467
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 468
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 468
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 469
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 469
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 470
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 470
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 471
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 471
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 472
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 472
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 473
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 473
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 474
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 474
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 475
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 475
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 476
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 476
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 477
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 477
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 478
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 478
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 479
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 479
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 480
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 480
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 481
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 481
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 482
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 482
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 483
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 483
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 484
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 484
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 485
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 485
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 486
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 486
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 487
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 487
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 488
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 488
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 489
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 489
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 490
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 490
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 491
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 491
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaatacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct a 91

<210> 492
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 492
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcacagaga atggtggatt 60
tccatacact gaaatgattg ttcatcta 88

<210> 493
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 493
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catcta 86

<210> 494
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 494
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 495
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 495
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 496
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 496
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 497
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 497
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 498
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 498
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcacagaga atggtggatt 60
tccatacact gaaatgattg ttcatcta 88

<210> 499
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 499
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 500
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 500
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 501
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 501
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 502
<211> 83
<212> DNA
<213> Nicotiana tabacum
<400> 502
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttagaatggt ggatttccat 60
acactgaaat gattgttcat cta 83

<210> 503
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 503
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 504
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 504
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatcta 89

<210> 505
<211> 85
<212> DNA
<213> Nicotiana tabacum
<400> 505
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacactg gtggatttcc 60
atacactgaa atgattgttc atcta 85

<210> 506
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 506
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 507
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 507
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 508
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 508
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 509
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 509
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 510
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 510
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 511
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 511
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 512
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 512
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 513
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 513
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 514
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 514
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 515
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 515
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 516
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 516
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 517
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 517
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 518
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 518
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 519
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 519
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 520
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 520
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 521
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 521
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 522
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 522
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 523
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 523
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 524
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 524
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 525
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 525
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 526
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 526
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 527
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 527
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 528
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 528
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 529
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 529
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 530
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 530
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 531
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 531
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 532
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 532
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 533
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 533
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 534
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 534
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 535
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 535
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 536
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 536
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagaatggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 537
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 537
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacatac agagactggt 60
ggatttccat acactgaaat gattgttcat cta 93

<210> 538
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 538
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 539
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 539
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 540
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 540
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 541
<211> 85
<212> DNA
<213> Nicotiana tabacum
<400> 541
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaagaatg gtggatttcc 60
atacactgaa atgattgttc atctt 85

<210> 542
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 542
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 543
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 543
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 544
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 544
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 545
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 545
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 546
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 546
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 547
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 547
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 548
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 548
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 549
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 549
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 550
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 550
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 551
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 551
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 552
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 552
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 553
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 553
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 554
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 554
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 555
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 555
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 556
<211> 85
<212> DNA
<213> Nicotiana tabacum
<400> 556
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaagaatg gtggatttcc 60
atacactgaa atgattgttc atctt 85

<210> 557
<211> 85
<212> DNA
<213> Nicotiana tabacum
<400> 557
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaagaatg gtggatttcc 60
atacactgaa atgattgttc atctt 85

<210> 558
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 558
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 559
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 559
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 560
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 560
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 561
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 561
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 562
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 562
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 563
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 563
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 564
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 564
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 565
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 565
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 566
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 566
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 567
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 567
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 568
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 568
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 569
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 569
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 570
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 570
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 571
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 571
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 572
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 572
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 573
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 573
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 574
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 574
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 575
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 575
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 576
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 576
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 577
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 577
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 578
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 578
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 579
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 579
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 580
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 580
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 581
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 581
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 582
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 582
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 583
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 583
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 584
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 584
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 585
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 585
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 586
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 586
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 587
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 587
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 588
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 588
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 589
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 589
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 590
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 590
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 591
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 591
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 592
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 592
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 593
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 593
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 594
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 594
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 595
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 595
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 596
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 596
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 597
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 597
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 598
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 598
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 599
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 599
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 600
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 600
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 601
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 601
tcagcaactt atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 602
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 602
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 603
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 603
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 604
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 604
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaaagaga atggtggatt 60
tccatacact gaaatgattg ttcatctt 88

<210> 605
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 605
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 606
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 606
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 607
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 607
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 608
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 608
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 609
<211> 84
<212> DNA
<213> Nicotiana tabacum
<400> 609
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcagaatgg tggatttcca 60
tacactgaaa tgattgttca tctt 84

<210> 610
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 610
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 611
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 611
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 612
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 612
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catctt 86

<210> 613
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 613
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catctt 86

<210> 614
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 614
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaaagaat ggtggatttc 60
catacactga aatgattgtt catctt 86

<210> 615
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 615
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 616
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 616
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaaagaat ggtggatttc 60
catacactga aatgattgtt catctt 86

<210> 617
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 617
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 618
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 618
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 619
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 619
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 620
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 620
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 621
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 621
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 622
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 622
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 623
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 623
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 624
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 624
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 625
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 625
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 626
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 626
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 627
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 627
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 628
<211> 86
<212> DNA
<213> Nicotiana tabacum
<400> 628
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcagagaat ggtggatttc 60
catacactga aatgattgtt catctt 86

<210> 629
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 629
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 630
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 630
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 631
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 631
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 632
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 632
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 633
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 633
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 634
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 634
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 635
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 635
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 636
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 636
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 637
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 637
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 638
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 638
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 639
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 639
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 640
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 640
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 641
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 641
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 642
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 642
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 643
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 643
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 644
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 644
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 645
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 645
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 646
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 646
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 647
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 647
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 648
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 648
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 649
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 649
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 650
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 650
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 651
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 651
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 652
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 652
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 653
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 653
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 654
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 654
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 655
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 655
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 656
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 656
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 657
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 657
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 658
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 658
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 659
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 659
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 660
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 660
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 661
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 661
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 662
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 662
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 663
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 663
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 664
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 664
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 665
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 665
tcagcaacat atgggaaggt tctgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 666
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 666
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 667
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 667
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 668
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 668
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacgaga atggtggatt 60
tccatacact gaaatgattg ttcatctt 88

<210> 669
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 669
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacgaga atggtggatt 60
tccatacact gaaatgattg ttcatctt 88

<210> 670
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 670
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaaagaga atggtggatt 60
tccatacact gaaatgattg ttcatctt 88

<210> 671
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 671
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaaagaga atggtggatt 60
tccatacact gaaatgattg ttcatctt 88

<210> 672
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 672
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 673
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 673
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 674
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 674
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 675
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 675
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 676
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 676
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 677
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 677
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 678
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 678
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 679
<211> 83
<212> DNA
<213> Nicotiana tabacum
<400> 679
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttagaatggt ggatttccat 60
acactgaaat gattgttcat ctt 83

<210> 680
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 680
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 681
<211> 83
<212> DNA
<213> Nicotiana tabacum
<400> 681
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttagaatggt ggatttccat 60
acactgaaat gattgttcat ctt 83

<210> 682
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 682
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 683
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 683
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 684
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 684
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 685
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 685
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 686
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 686
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 687
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 687
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 688
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 688
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 689
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 689
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 690
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 690
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 691
<211> 88
<212> DNA
<213> Nicotiana tabacum
<400> 691
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacaaga atggtggatt 60
tccatacact gaaatgattg ttcatctt 88

<210> 692
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 692
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 693
<211> 89
<212> DNA
<213> Nicotiana tabacum
<400> 693
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacagag aatggtggat 60
ttccatacac tgaaatgatt gttcatctt 89

<210> 694
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 694
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 695
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 695
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 696
<211> 91
<212> DNA
<213> Nicotiana tabacum
<400> 696
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacag agaatggtgg 60
atttccatac actgaaatga ttgttcatct t 91

<210> 697
<211> 85
<212> DNA
<213> Nicotiana tabacum
<400> 697
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaagaatg gtggatttcc 60
atacactgaa atgattgttc atctt 85

<210> 698
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 698
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 699
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 699
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 700
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 700
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 701
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 701
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 702
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 702
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 703
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 703
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 704
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 704
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 705
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 705
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 706
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 706
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 707
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 707
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 708
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 708
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 709
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 709
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 710
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 710
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 711
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 711
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 712
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 712
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 713
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 713
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 714
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 714
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 715
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 715
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 716
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 716
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 717
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 717
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 718
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 718
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 719
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 719
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 720
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 720
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 721
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 721
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 722
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 722
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 723
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 723
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 724
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 724
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 725
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 725
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 726
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 726
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 727
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 727
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 728
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 728
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

<210> 729
<211> 93
<212> DNA
<213> Nicotiana tabacum
<400> 729
tcagcaacat atgggaaggt tttgactttg gatggagcaa ttcaacacac agagaatggt 60
ggatttccat acactgaaat gattgttcat ctt 93

Claims (25)

(a) A first knockout mutant allele in the putrescine N-methyltransferase (PMT) 1a ( PMT1a) gene;
(b) a second knockout mutant allele in the PMT1b gene;
(c) a third knockout mutant allele in the PMT2 gene;
(d) a fourth knockout mutant allele in the PMT3 gene, wherein a wild type allele of the PMT3 gene encodes the amino acid sequence of SEQ ID NO: 14, and the fourth knockout mutant allele in the PMT3 gene comprises the nucleic acid sequence of SEQ ID NO: 630; and
(e) A fifth knockout mutant allele in the PMT4 gene.
A tobacco plant or part thereof comprising :
The tobacco plant or part thereof, wherein the tobacco plant, when grown and processed under equivalent conditions, produces leaves containing a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant that does not have the first knockout mutant allele, the second knockout mutant allele, the third knockout mutant allele, the fourth knockout mutant allele, and the fifth knockout mutant allele . 2. The tobacco plant or part thereof of claim 1, wherein a wild-type allele of the PMT1a gene encodes the polypeptide of SEQ ID NO: 12, a wild-type allele of the PMT1b gene encodes the polypeptide of SEQ ID NO: 11, a wild-type allele of the PMT2 gene encodes the polypeptide of SEQ ID NO: 13, and a wild-type allele of the PMT4 gene encodes the polypeptide of SEQ ID NO: 15. 3. The tobacco plant or part thereof of claim 1 or 2, wherein the tobacco plant is capable of producing leaves containing nicotine levels that are 90 % lower than the nicotine levels of leaves from a control tobacco plant that does not have the one or more mutant alleles when grown and processed under equivalent conditions. 4. The tobacco plant or part thereof according to any one of claims 1 to 3, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less than 90% of the total alkaloid level of leaves from the control tobacco plant when grown and processed under equivalent conditions. 5. The tobacco plant or part thereof of claim 4, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less than 40% of the total alkaloid level of leaves from the control tobacco plant when grown and processed under equivalent conditions. 6. The tobacco plant or part thereof of claim 1, wherein one or more of the first knockout mutant allele, the second knockout mutant allele, the third knockout mutant allele, or the fifth knockout mutant allele comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5 ' untranslated region ( UTR ) , a first exon, a first intron, a second exon, a second intron, a third exon, a 3'UTR, a terminator, and any combination thereof. 7. The tobacco plant or part thereof according to any one of claims 1 to 6, wherein one or more of the first knockout mutant allele, the second knockout mutant allele, the third knockout mutant allele, or the fifth knockout mutant allele comprise one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, and any combination thereof. One or more of the first knockout mutant allele, the second knockout mutant allele, the third knockout mutant allele, or the fifth knockout mutant allele are selected from the following:
8. The tobacco plant or part thereof according to any one of claims 1 to 7 , resulting in one or more of PMT protein truncation, non-translated PMT gene transcripts, non-functional PMT proteins, premature stop codons in the PMT gene, and any combination thereof. 9. The tobacco plant or part thereof according to any one of claims 1 to 8, wherein one or more of the first knockout mutant allele, the second knockout mutant allele, the third knockout mutant allele, or the fifth knockout mutant allele comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to a corresponding wild-type PMT gene sequence . 10. The tobacco plant or part thereof of any one of claims 1 to 9, which is homozygous for the first knockout mutant allele, the second knockout mutant allele, the third knockout mutant allele, the fourth knockout mutant allele, the fifth knockout mutant allele, or any combination thereof . 11. A tobacco plant or part thereof according to any one of claims 1 to 10 , wherein the tobacco plant is capable of producing leaves containing a nicotine level of less than 0.15% by dry weight. 12. The tobacco plant or part thereof of any one of claims 1 to 11 , wherein the tobacco plant is capable of producing leaves containing a total alkaloid level of less than 1% by dry weight. 13. The tobacco plant or part thereof according to any one of claims 1 to 12 , wherein said tobacco plant is capable of producing cured leaves containing a total TSNA level of from 2 to 0.05 ppm . A population of tobacco plants according to any one of claims 1 to 13 . A dried tobacco material derived from the tobacco plant according to any one of claims 1 to 13 . 16. The cured tobacco material of claim 15 , selected from the group consisting of flue-cured tobacco material , air-cured tobacco material , flame-cured tobacco material , and sun-cured tobacco material . 1. A dried tobacco material comprising tobacco leaves,
16. The cured tobacco material of claim 15 , wherein the tobacco leaves exhibit reduced mold infection compared to a control cured tobacco material derived from cultivar LA Burley 21. A tobacco blend comprising the dry tobacco material of claim 15 . 20. The tobacco blend of claim 18 , wherein the dry tobacco material comprises at least about 10 % by weight of the dry tobacco in the tobacco blend. 20. The tobacco blend of claim 18 , wherein the dry tobacco material comprises at least about 10 % by volume of the dry tobacco in the tobacco blend. A tobacco product comprising the dried tobacco material of claim 15 . 22. The tobacco product of claim 21, selected from the group consisting of cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. 22. The tobacco product of claim 21 , which is a smokeless tobacco product. 24. The tobacco product of claim 23, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff , and nasal snuff. 16. A reconstituted tobacco comprising the dried tobacco material of claim 15 .

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